Ann Lab Med.  2016 Jul;36(4):342-352. 10.3343/alm.2016.36.4.342.

Liquid Chromatography-Mass Spectrometry-Based In Vitro Metabolic Profiling Reveals Altered Enzyme Expressions in Eicosanoid Metabolism

• Lee SH
• Kim EJ
• Lee DH
• Lee WY
• Chung BC
• Seo HS
• Choi MH

• KMID: 2373554
• DOI: http://doi.org/10.3343/alm.2016.36.4.342

Abstract

BACKGROUND
Eicosanoids are metabolites of arachidonic acid that are rapidly biosynthesized and degraded during inflammation, and their metabolic changes reveal altered enzyme expression following drug treatment. We developed an eicosanoid profiling method and evaluated their changes on drug treatment.
METHODS
Simultaneous quantitative profiling of 32 eicosanoids in liver S9 fractions obtained from rabbits with carrageenan-induced inflammation was performed and validated by liquid chromatography-mass spectrometry coupled to anion-exchange solid-phase purification.
RESULTS
The limit of quantification for the devised method ranged from 0.5 to 20.0 ng/mg protein, and calibration linearity was achieved (R 2>0.99). The precision (% CV) and accuracy (% bias) ranged from 4.7 to 10.3% and 88.4 to 110.9%, respectively, and overall recoveries ranged from 58.0 to 105.3%. Our method was then applied and showed that epitestosterone treatment reduced the levels of all eicosanoids that were generated by cyclooxygenases and lipoxygenases.
CONCLUSIONS
Quantitative eicosanoid profiling combined with in vitro metabolic assays may be useful for evaluating metabolic changes affected by drugs during eicosanoid metabolism.

Keyword

Eicosanoids; Arachidonic acid; Liquid chromatography-mass spectrometry; Liver S9 fraction; Epitestosterone

MeSH Terms

Animals
Carrageenan/toxicity
*Chromatography, High Pressure Liquid/standards
Cytokines/blood
Disease Models, Animal
Eicosanoids/*analysis/metabolism/standards
Inflammation/etiology/metabolism
Male
Rabbits
Reference Standards
Solid Phase Extraction
*Tandem Mass Spectrometry/standards
Carrageenan
Cytokines
Eicosanoids

Figure

• 

  Fig. 1 Representative LC-SIM/MS chromatograms for the 32 eicosanoids analyzed in rabbit liver S9 fractions.Abbreviations: See Table 1; SIM, selected-ion monitoring.

• 

  Fig. 2 Quantification of eicosanoids in liver S9 fractions obtained from CGN-treated rabbits and control (untreated) rabbits. Differences in eicosanoid levels were examined for (A) COX-, (B) CYP ω-hydroxylase-, and (C) LOX-derived metabolites with 50 and 100 µM arachidonic acid. *P<0.05 and **P<0.01.Abbreviations: See Table 1; CGN, carrageenan; LOX, lipoxygenase.

• 

  Fig. 3 Effects of celecoxib and ibuprofen on eicosanoid production. Metabolite concentrations in the presence and absence of COX inhibitors (50 or 100µM) were measured. *P<0.05.Abbreviations: See Table 1; CGN, carrageenan.

• 

  Fig. 4 Changes in eicosanoid metabolism affected by epitestosterone in liver S9 fractions from CGN-induced rabbit models of inflammation. Different levels of arachidonic acid (50 or 100µM) were tested. Differences among eicosanoid levels after treatment with epitestosterone (50 or 100µM) were measured (n=8, each concentration). *P<0.05.Abbreviations: See Table 1; CYP, cytochrome P450; LOX, lipoxygenase; CGN, carrageenan.

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