Method Article

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

DOI:

10.3791/3399

⸱

November 17th, 2011

 ,  , 
1Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania , 2Department of Pharmacology, University of Pennsylvania

In This Article

Summary

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This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

Abstract

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The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.

While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.

Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

Protocol

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1. Standard and Internal Standard Mixes

  1. Before lipid extraction, one must prepare a standard mix (SM) and an internal standard mix (ISM). For the SM, aliquot the equivalent volume of 1 μg of each standard (25 total) listed in the reagent table below, using a calibrated syringe, into a 10 mL volumetric flask. Dry the standards under nitrogen and reconstitute in 10 mL of acetonitrile (ACN). The final concentration of the standard mix will be 100 pg/μL. Divide into 1 mL aliquots and store at -80°C until needed.
  2. From this standard mix, 6 standard dilutions can be made for a total of 7 calibration standards (1 pg/ μL, 2 pg/ μL, 5 pg/ μL, 10 pg/ μL, 20....

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Discussion

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The standards and internal standards used in this protocol provide a representation of a targeted lipidomics method. A Waters 2695 separation module and Thermo Fisher TSQ Quantum Ultra were used for the LC-MS analysis and the optimal parameter settings can be found in Tables 1 and 2. Additionally, this extraction protocol was designed for adherent cells, but can be modified for other cell types as well as other biological matrices including urine, blood, and tissue. Many lipid standards .......

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Disclosures

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No conflicts of interest declared.

Acknowledgements

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Most current bioanalytical methods available for the measurement of bioactive lipids are not as extensive as they do not include chiral normal phase chromatography or SID. Chiral normal phase LC is critical for the separation of enantiomers and for being able to distinguish between enzyme- or ROS-mediated metabolism. The use of SID ensures that human error or complications that arise during extraction or analysis are taken into account during quantification. These added components along with ECAPCI-MRM make this the most sensitive, selective method available for the analysis of bioactive lipids.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic-6,8,9,11,12,14,15-d7 acidCayman Chemical334250[2H7]-5-ox–TE Internal Standard
5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acidCayman Chemical334230[2H8]-5(S)-HETE Internal Standard
12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acidCayman Chemical334570[2H8]-12(S)-HETE Internal Standard
15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acidCayman Chemical334720[2H8]-15(S)-HETE Internal Standard
20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic-16,16,17,17,18,18-d6 acidCayman Chemical390030[2H6]-20-HETE Internal Standard
9S-hydroxy-10E,12Z-octadecadienoic-9,10,12,13-d4 acidCayman Chemical338410[2H4]-9(S)-HODE Internal Standard
13S-hydroxy-9Z,11E-octadecadienoic-9,10,12,13-d4 acidCayman Chemical338610[2H4]-13(S)-HODE Internal Standard
9α,11α,15S-trihydroxy-prosta-5Z,13E-dien-1-oic-17,17,18,18,19,19,20,20,20-d4 acidCayman Chemical316010[2H4]-PGF2α Internal Standard
9α,11α,15S-trihydroxy-(8β)-prosta-5Z,13E-dien-1-oic-3,3,4,4-d4 acidCayman Chemical316350[2H4]-8-iso-PGF2a Internal Standard
9α,11β.,15S-trihydroxy-prosta-5Z,13E-dien-1-oic-3,3,4,4-d4 acidCayman Chemical10008989[2H4]-11β-PGF2 Internal Standard
9α,15S-dihydroxy-11-oxo-prosta-5Z,13E-dien-1-oic-17,17,18,18,19,19,20,20,20-d4 acidCayman Chemical312010[2H4]-PGD2 Internal Standard
9-oxo-11α,15S-dihydroxy-prosta-5Z,13E-dien-1-oic-17,17,18,18,19,19,20,20,20-d4 acidCayman Chemical314010[2H4]-PGE2 Internal Standard
5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic-6,7,14,15-d4 acidCayman Chemical320110[2H4]-LTB4 Internal Standard
9α,11,15S-trihydroxy-thromba-5Z,13E-dien-1-oic-3,3,4,4-d4 acidCayman Chemical319030[2H4]-TxB2 Internal Standard
5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acidCayman Chemical342505-ox–TE Standard
12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acidCayman Chemical3458012-ox–TE Standard
15-oxo-5Z,8Z,11Z,13E-eicosatetraenoic acidCayman Chemical3473015-ox–TE Standard
5R-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acidCayman Chemical342255(R)-HETE Standard
5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acidCayman Chemical342305(S)-HETE Standard
8R-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acidCayman Chemical343508(R)-HETE Standard
8S-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acidCayman Chemical343608(S)-HETE Standard
11R-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acidCayman Chemical3450511(R)-HETE Standard
11S-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acidCayman Chemical3451011(S)-HETE Standard
12R-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acidCayman Chemical3456012(R)-HETE Standard
12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acidCayman Chemical3457012(S)-HETE Standard
15R-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acidCayman Chemical3471015(R)-HETE Standard
15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acidCayman Chemical3472015(S)-HETE Standard
20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acidCayman Chemical9003020-HETE Standard
9R-hydroxy-10E,12Z-octadecadienoic acidCayman Chemical384059(R)-HODE Standard
9S-hydroxy-10E,12Z-octadecadienoic acidCayman Chemical384109(S)-HODE Standard
13R-hydroxy-9Z,11E-octadecadienoic acidCayman Chemical3860513(R)-HODE Standard
13S-hydroxy-9Z,11E-octadecadienoic acidCayman Chemical3861013(S)-HODE Standard
9α,11α,15S-trihydroxy-prosta-5Z,13E-dien-1-oic acidCayman Chemical16010PGF2α Standard
9α,11α,15S-trihydroxy-(8β)-prosta-5Z,13E-dien-1-oic acidCayman Chemical163508-iso-PGF2α Standard
9α,11β,15S-trihydroxy-prosta-5Z,13E-dien-1-oic acidCayman Chemical1652011β-PGF2 Standard
9α,15S-dihydroxy-11-oxo-prosta-5Z,13E-dien-1-oic acidCayman Chemical12010PGD2 Standard
9-oxo-11α,15S-dihydroxy-(8β)-prosta-5Z,13E-dien-1-oic acidCayman Chemical143508-iso-PGE2 Standard
9-oxo-11α,15S-dihydroxy-prosta-5Z,13E-dien-1-oic acidCayman Chemical14010PGE2 Standard
5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic acidCayman Chemical20110LTB4 Standard
9α,11,15S-trihydroxythromba-5Z,13E-dien-1-oic acidCayman Chemical19030TxB2 Standard
Phosphate Buffered SalineGIBCO, by Life Technologies14190
Diethyl EtherSigma-Aldrich346136
DichloromethaneAcros Organics61030-1000anhydrous
N,N-diisopropylethyl amineSigma-Aldrich387649
Pentafluorylbenzyl bromideSigma-Aldrich101052
Hydrochloric AcidSigma-Aldrich320331
Potassium HydroxideFluka00650
AcetonitrileFisher ScientificA996-4
MethanolFisher ScientificA454-4
ChloroformFisher Scientific366927
HexaneFisher ScientificH303-4
IsopropanolFisher ScientificA464-4
EthanolDecon Laboratories2716
WaterFisher ScientificW7-4
Pasteur PipetsFisher Scientific13-678-200
10 mL Glass Centrifuge TubesKimble Chase73785-10Screw cap
Phenolic Screw CapsKimble Chase73802-13415
Chiralcel ADH ColumnChiral Technologies19325
HPLC vialsWaters60000751CV
HPLC insertsWatersWAT094171

References

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  1. Needleman, P., Turk, J., Jakschik, B. A., Morrison, A. R., Lefkowith, J. B. Arachidonic acid metabolism. Annu. Rev. Biochem. 55, 69-102 (1986).
  2. Nikolaev, V., Reddanna, P., Whelan, J., Hildenbrandt, G., Reddy, C. C. Stereochemical nature of the products....

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Tags

Electron Capture DerivatizationChiral Normal Phase HPLCMultiple Reaction MonitoringBioactive Lipid ExtractionLiquid Liquid ExtractionArachidonic Acid MetabolitesLinoleic Acid MetabolitesEnantiomer Separation

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