Eicosanoids Richard Harkewicz

LIPID MAPS LIPID MAPS Lipidomics Workshop April 18, 2009 TM www.lipidmaps.org Eicosanoids Richard Harkewicz

Departments of Pharmacology, Chemistry and Biochemistry The University of California, San Diego La Jolla, CA

Other LIPID MAPS Eicosanoid Core Members: Aaron Armando Matthew Buczynski Edward A. Dennis Alexander Andreyev Darren Dumlao (Core Director) Joshua Brooks Oswald Quehenberger 1 Outline A) Brief description of eicosanoids B) Sample preparation/extraction C) Analytical methodology: LC-MS non-enzymatic? D) Library of eicosanoid standards E) Chiral chromatography – enzymatic or

F) GC-MS methodology for free fatty acids G) DIMPLES/MS: A stable isotope substrate labeling strategy enabling the search for novel eicosanoids H) Comparison of LIPID MAPS eicosanoid approach with others in literature I) Future plans contact: [email protected] 2 stimulation release of (LPS or phospholipase A2 membrane phospholipid (outside Kdo-Lipid A) (PLA2) the2 cell) (R = choline or ethanolamine) O (inside (PLA2) R O P O CH2 the cell) OH CH O arachidonate COOH H2C O sterate (20:4) (18:0)

free eicosatetraenoic acid (from the Greek eicosa for 20) are derived fromAKA arachidonicarachidonic acid acid and(20:4, act n=6) in autocrine and paracrine Eicosanoidsfashion (signal at– powerful or immediately mediators ad involved in inflammation that

Once they are made, they are quickly secreted from the cell jacent to their site of synthesis) Transcription factor: Can also enter cell’s nucleus, binding and activating nuclear receptors Sometimes referred to as autocoids or local hormones 3 HO COOH COOH (vasoconstriction, O COOH smooth muscle O TXA O contraction) HO OH 2 HO OH COOH PGF2α

O HO OH PGD2 (vasodilation, OH PGI2 hypotension)

COX OH OH OOH COOH COOH COOH LTB4 (vasodilation, leukocyte OH 5-LO 5-HpETE adhesion, chemotaxis) AA COOH

CONHCH2COOH 15-LO S LTD NH2 4

OH OH COOH COOH COOH HO HO (vasodilation, chemotaxis, OH OOH 15-HpETE OH LXA4 LXB leukocyte activation) 4 4 sample preparation and extraction 0 h 24 h 48 h LC-MS Solid phase analysis Plate LPS or Collect extraction Medium Cells Kdo2-Lipid A [100 ng/ml]

1. Solid phase extraction (Phenomenex® Strata-X 8B-S100-UBJ) 1. Spike medium 1. Plate cells with deuterated 2. Prewash SPE column 24 h internal standards with 2 ml MeOH 2. Stimulate (10 ng/100 μl) then 2 ml H2O (LPS or Kdo) 3. Apply sample to column 2. Add EtOH to 24 h then wash with 2 ml 10% obtain 10% EtOH MeOH 3. Remove medium medium solution (≈2 ml per well) 4. Elute eicosanoids with (≈2e6 cells per well) 3. Centrifuge 5 min 1 ml MeOH @ 3000 rpm 5. Dry under vacuum and resuspend in 100 μl HPLC solvent A 5 column Solvent A RP HPLC Gradient 80 Vydac® 201TP52 H20/ACN/formic acid: 2.1 mm X 250 mm 63/37/0.02 60 Solvent B Flow rate 300 μl/min ACN/IPA: 50/50 40 solvent A solvent B Solvent B (%) 20

LC pump 0 0 5 10 15 20 25 Time (min)

auto RPLC C column ABI Analyst sampler 18 collision software gas (N2)

()ESI CEM detector Q1 mass q2 Q3 linear trap analyzer collision mass cell analyzer Applied Biosystems® 4000 QTrap Quadrupole mass spectrometer 6 7

T M +

)

( A A isolate product isolate

great S/N & comprehensive MRM pair:

fragment precursor

203)

335/

4 (

T L Q1 q2 Q3 Applied Biosystems 1.5 Analyst HETEs acids)

“scheduled MRM” allowing >1000 MRM pairs

)

2 Time, min /

isolate precursor 5

3 )

(

2 (hydroxyeicosatertraenoic

351

(

P )

1 /

(

6 2

G onitoring 9

36 2 (

M X T 2 4 6 8 10 12 14 16

eaction Mixture R LC/MS - MRM Eicosanoid Standard

ultiple 75% 50% 25%

100% Rel. Intensity Rel. M “specialized” of MSMS form COOH The HETEs OH

COOH CH3 HO 11(s) HETE 8(s) HETE (319/155) (319/167) CH3

5(s) HETE (319/115) OH

COOH COOH

CH3 CH3 12(s) HETE OH (319/179)

15(s) HETE (319/113) COOH

CH3 OH

10.8 11.0 11.2 11.4 11.6 11.8 Time, min 8 Library of Eicosanoid Standards (>200) (http://www.lipidmaps.org) Provides:

Chemical structure in ChemDraw® format

LC and MS protocols

MSMS fragmentation spectra

LC retention times for given set of conditions

Web-link to Cayman Chemical for each eicosanoid

The eicosanoid library provides information from which comprehensive methods

T can be created and used to survey ei M 9

cosanoid release from stimulated cells use of deuterium labeled internal standards allows absolute quantitation for a number of eicosanoids

D D O - O - COO COO D D

HO HO OH OH - PGE -d4 [M-H]- = 355 PGE2 [M-H] = 351 2

example of internal standard calibration curves used for quantitation

PGE 12,14 2 15d- D PGJ2 5-HETE MRM: 351/189 MRM: 315/271 MRM: 319/115 100 100 100 r ² = 0.9995 r ² = 0.9975 10 10 10 r ² = 0.9993 1 1 1 0.1

MRM Ratio 0.1 0.1 0.01

0.01 0.01 0.001 0.1 1 10 100 1000 0.1 1 10 100 1000 0.1 1 10 100 1000 Analyte (ng) 10 11 l/min μ Solvent B EtOH : 100 Flow rate 500 Time (min) AD-H NP HPLC Gradient NP HPLC ® 0/formic acid: 2 column Solvent A Chiralpak 4.6 mm X 250 mm 96/4/0.08/0.02 010203040

50 40 30 20 10 Solvent B (%) B Solvent hexane/EtOH/H STD MRM: 15(S) HETE 319/113 10.27 10.24 Time, min Time, Time, min Time, RAW cells RAW 9.35 9.32 -Lipid A stimulated STD 2 8.6 9.4 10.2 11.0 8.6 9.4 10.2 11.0 15(R) HETE vs. nonenzymatic origin Kdo STD 11(S) HETE MRM: 319/167 9.34 8.87 8.85 Time, min Time, Time, min Time, RAW cells RAW chiral chromatography aids in determining enzymatic ionization (APCI) used -Lipid A stimulated STD 2

atmospheric pressure chemical Kdo 11(R) HETE 7.6 8.4 9.2 10.0 7.6 8.4 9.2 10.0 12

pp. 123-129 anions. - Produces intact molecular

GC-MS analysis of free fatty acids GC-MS analysis of

Essent. Fatty Acids 79

analysis of essential fatty acids in macrophages Quehenberger et al. (2008) Prostaglandins Leukot. Agilent 6890N gas chromatograph Agilent 5973 single quadrupole mass analyzer Selected ion monitoring (SIM) mode monitoring the [M-H] Pentafluorobenzyl (PFB) derivatives of the free fatty acids are formed and negative chemical ionization employed. anions - optimal for quantitation (using deuterium labeled standards). Are biologically significant eicosanoids being overlooked? Are there species for which we have no prior knowledge of or expectation of their presence and,

To address theserequired concerns for atheir mass detection? spectral based stable isotope labeling strategy hence,has been no available developed MRM pairs

DIMPLES/MS: Diverse Isotope Metabolic Profiling of Labeled Exogenous Substrates using Mass Spectrometry Harkewicz et al. (2007) J. Biol. Chem. 282 pp. 2899-2910 Incubation of cells in medium supplemented with deuterium-labeled arachidonic acid (AA-d8)

D D D D COOH COOH

AA D D D D AA-d8 13 uptake and incorporation of AA-d8 by cells

0 h 24 h 48 h LC-UV-MS solid phase analysis plate Kdo2-Lipid A collect extraction cells [100 ng/ml] medium add AA-d8 [12.5 μg/ml, 40µM ] exchange of the PL’s endogenous sn2 fatty acids for the exogenous AA-d8

100 % (R = choline or ethanolamine) O AA-d8 AA

R O P O CH2 60 % OH CH O arachidonate

H2C O sterate

20 % AA-d8 Remaining in Media (%) AA-d8 Remaining 0 5 10 15 20 AA-d8 Incubation Time (h) 14 so as not to be a AA/AA-d8 mass spectral doublet targeted analysis full-scan MS indicative(1) AA of origin eicosanoid: 10% serum (fetal calf) 2) upregulated by Kdo AA-d8 supplemented 357

D D D D [(PGD + d6) - H]- 2 COOH

351 - [PGD2 -H] D D D D AA-d8

358 - COX [(PGD2+ d5) - H] - [(PGD2+ d7) - H] + PGD-synthase Rel. Intensity (%) Rel. Intensity 356

OH D D D D 352 COOH 359 355 353 O D D HO D PGD2+ d7 351 353 355 357 359 m/z, amu 15 observed production of a class of dihomoprostaglandins Harkewicz et al. (2007) J. Biol. Chem. 282 pp. 2899-2910

Kdo – Lipid A stimulated RAW cells HO dihomo-PGD2 2 O dihomo-PGE2 Control (NO AA-d8) COOH COOH

AA-d8 supplemented O HO OH OH 385 COOH 385 - [(M+d6) - H]- [(M+d6) - H] - m/z = 331 [M-H] [M-H]-

379 adrenic acid (22:4 n-6) 379 339 384 386 100% 386 384 380 380 381 383 387 [(22:4 n-6) + d8 – H]- 381 383 387

HO dihomo-PGF2α dihomo-11(R)HETE COOH COOH

HO 389 HO - 347 [(M+d8) - H]- OH [(M+d8) - H] [M-H]- 354 355 [M-H]- [(22:4 n-6) – H]- 388 381 353 390 348 387 356 331 352 357 382 383 386 349

12,14 12,14 dihomo-∆ -PGJ dihomo-15d-Δ -PGD2 Rel. Intensity 338 2 HO COOH COOH

O OH 367 O 367 340 [(M+d6) - H]- 332 337 [M-H]- [(M+d6) - H]- [M-H]- 333 361 361 366 368 368 366 362 362 365 369 363 365 369 331 334 337 363 m/z, amu 16 another approach to eicosanoid lipidomics and the search for novel eicosanoids Serhan Lab developing theoreti based on virtual UV absorption, tandem mass spectrometry and LC retention times for identifying potential eicosanoids .

Y. Lu et al.cal (2005) databases J. Lipid andRes. 46algorithmspp. 790-802

select spectra and chromatograms of MS. MS/MS, and UV LM: lipid mediators acquired via LC-UV-MS/MS for LMs in samples CRT: chromatographic direct searches towards compounds having expected or unexpected molecular ions retention time YES NO match standard UV match UV of theoretical structures in database in databases?

YES NO match MS/MS of theoretical structures in database match standard MS/MS in databases? match CRTs of theoretical structures in database YES match standard CTRs NO identified isomers of in databases? the LM standards potentially novel YES LMs proposed LM identified 17 Future Plans

Continue to expand eicosanoid library

Incorporate UV detection and analyses into eicosanoid surveys

Expand search for novel eicosanoids

Similar studies with Ω-3 fatty acid supplementation EPA (20:5 n-3) and DHA (22:6 n-3)

18 LIPID MAPS

TM www.lipidmaps.org