Cellulaire vetextractie voor gerichte stabiele isotopen Verdunning vloeistofchromatografie-massaspectrometrie Analyse
Summary
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
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
Discussion
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 …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic-6,8,9,11,12,14,15-d7 acid | Cayman Chemical | 334250 | [2H7]-5-oxoETE Internal Standard |
| 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid | Cayman Chemical | 334230 | [2H8]-5(S)-HETE Internal Standard |
| 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid | Cayman Chemical | 334570 | [2H8]-12(S)-HETE Internal Standard |
| 15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid | Cayman Chemical | 334720 | [2H8]-15(S)-HETE Internal Standard |
| 20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic-16,16,17,17,18,18-d6 acid | Cayman Chemical | 390030 | [2H6]-20-HETE Internal Standard |
| 9S-hydroxy-10E,12Z-octadecadienoic-9,10,12,13-d4 acid | Cayman Chemical | 338410 | [2H4]-9(S)-HODE Internal Standard |
| 13S-hydroxy-9Z,11E-octadecadienoic-9,10,12,13-d4 acid | Cayman Chemical | 338610 | [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 acid | Cayman Chemical | 316010 | [2H4]-PGF2α Internal Standard |
| 9α,11α,15S-trihydroxy-(8β)-prosta-5Z,13E-dien-1-oic-3,3,4,4-d4 acid | Cayman Chemical | 316350 | [2H4]-8-iso-PGF2a Internal Standard |
| 9α,11β.,15S-trihydroxy-prosta-5Z,13E-dien-1-oic-3,3,4,4-d4 acid | Cayman Chemical | 10008989 | [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 acid | Cayman Chemical | 312010 | [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 acid | Cayman Chemical | 314010 | [2H4]-PGE2 Internal Standard |
| 5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic-6,7,14,15-d4 acid | Cayman Chemical | 320110 | [2H4]-LTB4 Internal Standard |
| 9α,11,15S-trihydroxy-thromba-5Z,13E-dien-1-oic-3,3,4,4-d4 acid | Cayman Chemical | 319030 | [2H4]-TxB2 Internal Standard |
| 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid | Cayman Chemical | 34250 | 5-oxoETE Standard |
| 12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acid | Cayman Chemical | 34580 | 12-oxoETE Standard |
| 15-oxo-5Z,8Z,11Z,13E-eicosatetraenoic acid | Cayman Chemical | 34730 | 15-oxoETE Standard |
| 5R-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid | Cayman Chemical | 34225 | 5(R)-HETE Standard |
| 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid | Cayman Chemical | 34230 | 5(S)-HETE Standard |
| 8R-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid | Cayman Chemical | 34350 | 8(R)-HETE Standard |
| 8S-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid | Cayman Chemical | 34360 | 8(S)-HETE Standard |
| 11R-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid | Cayman Chemical | 34505 | 11(R)-HETE Standard |
| 11S-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid | Cayman Chemical | 34510 | 11(S)-HETE Standard |
| 12R-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid | Cayman Chemical | 34560 | 12(R)-HETE Standard |
| 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid | Cayman Chemical | 34570 | 12(S)-HETE Standard |
| 15R-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid | Cayman Chemical | 34710 | 15(R)-HETE Standard |
| 15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid | Cayman Chemical | 34720 | 15(S)-HETE Standard |
| 20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid | Cayman Chemical | 90030 | 20-HETE Standard |
| 9R-hydroxy-10E,12Z-octadecadienoic acid | Cayman Chemical | 38405 | 9(R)-HODE Standard |
| 9S-hydroxy-10E,12Z-octadecadienoic acid | Cayman Chemical | 38410 | 9(S)-HODE Standard |
| 13R-hydroxy-9Z,11E-octadecadienoic acid | Cayman Chemical | 38605 | 13(R)-HODE Standard |
| 13S-hydroxy-9Z,11E-octadecadienoic acid | Cayman Chemical | 38610 | 13(S)-HODE Standard |
| 9α,11α,15S-trihydroxy-prosta-5Z,13E-dien-1-oic acid | Cayman Chemical | 16010 | PGF2α Standard |
| 9α,11α,15S-trihydroxy-(8β)-prosta-5Z,13E-dien-1-oic acid | Cayman Chemical | 16350 | 8-iso-PGF2α Standard |
| 9α,11β,15S-trihydroxy-prosta-5Z,13E-dien-1-oic acid | Cayman Chemical | 16520 | 11β-PGF2 Standard |
| 9α,15S-dihydroxy-11-oxo-prosta-5Z,13E-dien-1-oic acid | Cayman Chemical | 12010 | PGD2 Standard |
| 9-oxo-11α,15S-dihydroxy-(8β)-prosta-5Z,13E-dien-1-oic acid | Cayman Chemical | 14350 | 8-iso-PGE2 Standard |
| 9-oxo-11α,15S-dihydroxy-prosta-5Z,13E-dien-1-oic acid | Cayman Chemical | 14010 | PGE2 Standard |
| 5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic acid | Cayman Chemical | 20110 | LTB4 Standard |
| 9α,11,15S-trihydroxythromba-5Z,13E-dien-1-oic acid | Cayman Chemical | 19030 | TxB2 Standard |
| Phosphate Buffered Saline | Gibco | 14190 | |
| Diethyl Ether | Sigma | 346136 | |
| Dichloromethane | Acros | 61030-1000 | anhydrous |
| N,N-diisopropylethyl amine | Sigma | 387649 | |
| Pentafluorylbenzyl bromide | Sigma | 101052 | |
| Hydrochloric Acid | Sigma | 320331 | |
| Potassium Hydroxide | Fluka | 00650 | |
| Acetonitrile | Fisher | A996-4 | |
| Methanol | Fisher | A454-4 | |
| Chloroform | Fisher | 366927 | |
| Hexane | Fisher | H303-4 | |
| Isopropanol | Fisher | A464-4 | |
| Ethanol | Decon Labs | 2716 | |
| Water | Fisher | W7-4 | |
| Pasteur Pipets | Fisher | 13-678-200 | |
| 10 mL Glass Centrifuge Tubes | Kimble | 73785-10 | Screw cap |
| Phenolic Screw Caps | Kimble | 73802-13415 | |
| Chiralcel ADH Column | Chiral Technologies | 19325 | |
| HPLC vials | Waters | 60000751CV | |
| HPLC inserts | Waters | WAT094171 |
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