Lipids

Sample Preparation 

• Tissue/cell samples should be frozen in liquid nitrogen immediately after collection.
• Serum or EDTA plasma should be removed from the red cells as soon as possible after collection and then frozen.
• Please label all tubes clearly and legibly.
• All samples should be stored at -70°C until shipped.
• Heparin is not acceptable as an anticoagulant for lipid profiles.
• Samples with gross hemolysis or lipemia may yield false results.
• Complete the order submission form online, make a copy, and place it in the container with your samples enclosed in a plastic bag.

Required Volumes

Plasma Lipid Profiles

Total plasma cholesterol and triglyceride are measured by standard enzymatic assays.  HDL cholesterol is measured with the enzymatic method after precipitation of VLDL and LDL using polyethylene glycol reagent (PEG).  From these data LDL cholesterol is calculated using the Friedewald equation, as long as triglyceride levels are below 400 mg/dl. Investigators may request a total plasma lipid profile or specific plasma lipid measurements.

Plasma Enzymatic Free Fatty Acids (FFA) 

Plasma FFAs are analyzed with a commercially available enzymatic kit from Fujifilm Healthcare Solutions.

Plasma Free Fatty Acid GC Analysis

Free fatty acids are extracted from the plasma using heptane/isopropanol (30:70) (1) and separated from other lipids by thin layer chromatography on silica gel plates developed in petroleum ether/ethyl ether/acetic acid (80:20:1 v/v/v).  The free fatty acid band is visualized using rhodamine 6G (0.1% in ethanol), scraped from the TLC plate, and methylated with BF₃/methanol(2) without elution from the silica gel. 

The fatty acid methyl esters are analyzed by gas chromatography using an Agilent 7890 gas chromatograph equipped with flame ionization detector and a capillary column (SP2380, 0.25 mm x 30 m, 0.20 µm film, Supelco, Bellefonte, PA).  Helium is used as a carrier gas.  The oven temperature is programmed from 160 °C to 230 °C at 4 °C/min.  Fatty acid methyl esters are identified by comparing the retention times to those of known standards.  Inclusion of a pentadecanoic acid (15:0) internal standard permits quantitation of the amount of FFA in the sample.

References

1. Ko, H., and Royer, M. E.  1974. A gas-liquid chromatographic assay for plasma free fatty acids.  J. Chrom.  88:  253-263.
2. Morrison, W. R., and Smith, L. M.  1964.  Preparation of fatty acid methyl esters and dimethylacetal from lipids with boron trifluoride-methanol.  J. Lipid Res.  5:  600-608.

Tissue/Cell Lipid GC Analysis

Lipids are extracted using the method of Folch-Lees (1).  The extracts are filtered and lipids recovered in the chloroform phase.  Individual lipid classes are separated by thin layer chromatography using Silica Gel 60 A plates developed in petroleum ether, ethyl ether, acetic acid (80:20:1) and visualized by rhodamine 6G.   Phospholipids, diglycerides,  triglycerides and cholesteryl esters are scraped from the plates and methylated using BF₃ /methanol as described by Morrison and Smith (2).   The methylated fatty acids are extracted and analyzed by gas chromatography.   Gas chromatographic analyses are carried out on an Agilent 7890A gas chromatograph equipped with flame ionization detectors and a capillary column (SP2380, 0.25 mm x 30 m, 0.20 µm film, Supelco, Bellefonte, PA).  Helium is used as the carrier gas.  The oven temperature is programmed from 160 °C to 230 °C at 4 °C/min.  Fatty acid methyl esters are identified by comparing the retention times to those of known standards.  Inclusion of lipid standards with odd chain fatty acids permits quantitation of the amount of lipid in the sample.  Dipentadecanoyl phosphatidylcholine (C15:0), diheptadecanoin (C17:0), trieicosenoin (C20:1), and cholesteryl eicosenoate (C20:1) are used as standards. 

References

1. J. Folch, M. Lees, and G.H. Sloane-Stanley. 1957.  A simple method for the isolation and purification of total lipides from animal tissues.  J. Biol. Chem. 226: 497-509. 
2. R. Morrison and L.M. Smith. 1964.  Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol.  J. Lipid Res. 5: 600-608.

Tissue/Cell Cholesterol GC Analysis

Unesterified Cholesterol

Internal standard is added to a portion of the lipid extract, concentrated under nitrogen and then solubilized in hexane to inject onto the gas chromatograph.

Total Cholesterol

Internal standard (5-a-cholestane) is added to a portion of the lipid extract and then saponified at 80 C in 1 N KOH in 90% methanol for 1 hour.  The nonsaponifiable sterol is extracted into hexane, concentrated under nitrogen, and then solubilized in carbon disulfide to inject onto the gas chromatograph.

Samples are analyzed on an Agilent 7890A gas chromatograph equipped with an HP-50+ column (Agilent) and a flame ionization detector. The oven temperature is 260-280° C and helium is used as the carrier gas.

Adapted from: 

Rudel, L.L., K. Kelley, J.K. Sawyer, R. Shah, and M.D. Wilson.  1998.  Dietary monounsaturated fatty acids promote aortic atherosclerosis in LDL receptor-null, human apoB100-overexpressing transgenic mice. Aterioscler.Thromb. Vasc. Biol. 18: 1818-1827. 

Fast Protein Liquid Chromatography

Lipoprotein fractions are separated from 100 microliters of plasma (or serum) by gel filtration column chromatography.  Approximately 70 fractions (0.25 ml) are collected and the amount of triglyceride and cholesterol in each fraction is determined using microtiter plate, enzyme-based assays.   Profiles of triglyceride and cholesterol are constructed.