Eicosanoid determination - Harkewitz et al.

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Contents

Abstract

Harkewitz et al. developed a sensitive MS analysis to survey eicosanoid release from endotoxin-stimulated RAW 264.7 macrophage-like cells detecting over 70 diverse eiconanoids and eicosanoid metabolites. Therefor, they investigated four cell sample types: Kdo2-Lipid A treated (100 ng/ml) with (12,5 μg/ml; 24 h) or without AA-d8 supplementation and non-treated with or without AA-d8 supplementation. Operating this strategy, new and unexpected AA metabolites generated by the cells were observed including a series of 1a,1b-dihomologue prostaglandins, products of adrenic acid, resulting from the two-carbon elongation of AA. Thus, a comprehensive library of various eicosanoids was compiled and supposably this protocol can be extended to other labeled exogenous substrates and their metabolites too.

Analysed Matrices

mouse macrophage like cells

Analytes

  • 75 eicosanoids:

12(S)HHTrE; AA; AA-d8; 15d-∆12,14PGJ2; 5-OxoETE; 12-OxoETE; 15-OxoETE; 20-HETE; 5(R)HETE; 5(S)HETE; 8(R)HETE; 8(S)HETE; 11(R)HETE; 11(S)HETE; 12(R)HETE; 12(S)HETE; 15(R)HETE; 15(S)HETE; ±5,6-EpETrE; ±8,9-EpETrE; ±11,12-EpETrE; ±14,15-EpETrE; tetranor PGEM; 5(S)HETE-d8; tetranor PGFM; PGA2; PGB2; PGJ2; ∆12-PGJ2; dhk-PGA2; bicyclo-PGE2; 5(S)6(R)DiHETE; 5(S)6(S)DiHETE;5(S)15(S)DiHETE; 8(S)15(S)DiHETE; LTB4; 5(S)HpETE; 12(S)HpETE; 15(S)HpETE; ±5,6-DiHETrE; ±8,9-DiHETrE;±11,12-DiHETrE; ±14,15-DiHETrE; 2,3-dinor TXB4;15-keto PGE2;PGK2; PGD2; PGE2; PGH2; dhk-PGD2; dhk-PGE2;15-keto PGF2a; 5(S)6(R)15(S)LXA4; 5(S)6(S)15(S)LXA4;5(S)14(R)15(S)LXB4; PGF2a; 11b-PGF2a; PGF2b; dhk-PGF2a; PGD2-d4; PGE2-d4; PGF2a-d4; PGG2; 19(R)hydroxy PGE2; 20-hydroxy PGE2; 6-keto PGE1; 11-dehydro TXB2; TXB2; 6-keto PGF1a; diketo-dihydro PGF1a; PGD2-EA; PGE2-EA; PGF2a-EA; LTE4; LTC4

Sample Preparation

  • collection of cultured medium
  • centrifugation (3 min, 1000 rpm)
  • supplementation with 10 % MeOH

SPE

column: Strata-X (60 mg, 3 ml, phenomenex)

equilibration: 2 ml MeOH, 2 ml H2O

loading: 2 ml H2O/MeOH (90/10)

eluation: 1 ml MeOH (100 %)

  • evaporation of solvent
  • addition of 100 μl LC-buffer A

Analytical Method

HPLC-MS-MS

HPLC (Shimadzu LC-10AD)

columns: reversed phase C-18 (Vydac, 2.1*250 mm, 5 μl); guard column (Vydac); solvent A: H2O/CH3CN/HCOOH (63/37/0.02); solvent B: CH3CN/iPrOH (50/50); gradient: A/B = 100/0 (0 min) A/B = 80/20 (6 min) A/B = 45/55 (6,5 -11min) A/B = 28/72 (11,5 -16 min) A/B = 100/0 (18 -25 min); flow rate: 0.3 ml/min (A and B); injection volume: 10 μl, 30 μl

HPLC (chiral)

columns: normal phase (Chiralpak AD-H); guard column (Chiralpak AD-H); buffer A: Hex/EtOH/H2O/HCOOH (96/4/0.08/0.02); buffer B: EtOH (100 %);

gradient: A/B = 100/0 (0 min) A/B = 90/10 (13 min) A/B = 75/25 (15 -25 min) A/B = 100/0 (27 -42 min)

MS (Applied Bioscience 4000 QTRAP)

hybrid triple quadrupole; linear ion trap; Turbo V ion source; negative electrospray mode (CI for chiral chromatography); scan modi: Q1, MS2, MRM

QTRAP: CUR: 10 psi; GS1: 40 psi; GS2: 40 psi (60 psi for chiral chromatography); IS: -4200 V; collisional activated dissociation: HIGH (-20 to -40 V); T: 525 °C (400 °C for chiral chromatography); ihe: ON; DP: -30 V (-60 V for chiral chromatography); EP: -15 V; CXP: -10 V

Method Validation

LOD

n.d.

LOQ

n.d.

Linearity

n.d.

Internal Standard (IS)

deutered metabolites resulting from AA-d8-supplementation (AA-d8; 5(S)HETE-d8; PGD2-d4; PGE2-d4; PGF2a-d4)

Reference

  • R. Harkewitz, E. Fahy, A. Andreyev, E. A. Dennis J. Biolog. Chem. 2007, 282, 2899-2910.

Departments of Pharmacology, Chemistry, and Biochemistry; University of California; San Diego; 9500 Gilman Drive; La Jolla; CA 92093-0601.

Tel.: 858-534-3055; Fax: 858-534-7390; e-mail: edennis@ucsd.edu.

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