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

Original Article Laboratory Informatics Ann Lab Med 2016;36:342-352 http://dx.doi.org/10.3343/alm.2016.36.4.342 ISSN 2234-3806 • eISSN 2234-3814 Liquid Chromatography-Mass Spectrometry-Based In Vitro Metabolic Profiling Reveals Altered Enzyme Expressions in Eicosanoid Metabolism Su Hyeon Lee, M.S.1,2, Eung Ju Kim, M.D.3, Dong-Hyoung Lee, Ph.D.1, Won-Yong Lee, Ph.D.2, Bong Chul Chung, Ph.D.1, Hong Seog Seo, M.D.3, and Man Ho Choi, Ph.D.1 Materials and Life Science Research Division1, Korea Institute of Science and Technology, Seoul; Department of Chemistry2, Yonsei University, Seoul; Cardiovascular Center3, Korea University Guro Hospital, Seoul, Korea 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. Received: November 2, 2015 Revision received: December 8, 2015 Accepted: March 4, 2016 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. Corresponding author: Man Ho Choi Materials and Life Science Research Division, KIST, 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Korea Tel: +82-2-958-5081 Fax: +82-2-958-5059 E-mail: 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. Co-corresponding author: Hong Seog Seo Cardiovascular Center, Korea University Guro Hospital, 148 Gurodong-ro, Guro-gu, Seoul 08308, Korea Tel: +82-2-2626-3018 Fax: +82-2-863-1109 E-mail: Conclusions: Quantitative eicosanoid profiling combined with in vitro metabolic assays may be useful for evaluating metabolic changes affected by drugs during eicosanoid metabolism. © The Korean Society for Laboratory Medicine Key Words: Eicosanoids, Arachidonic acid, Liquid chromatography-mass spectrometry, Liver S9 fraction, Epitestosterone INTRODUCTION Signaling lipids in inflammation control important cellular processes, and an imbalance in metabolic homeostasis may contribute to the pathogenesis of human diseases such as cardiovascular and neurodegenerative diseases [1-3]. The chronic inflammatory response is partly mediated by lipid peroxidation, uncontrolled proliferation of immune-related cells, and activation of the innate immune system [4], whereas activation of the acute inflammatory response alters cytochrome P450 (CYP) ex- 342  www.annlabmed.org This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. pression and eicosanoid metabolism [5]. Lipid profiling can be used to map the entire spectrum of lipids in biological systems to identify changes in lipid biosynthesis and metabolism that contribute to inflammatory processes [6-8]. Eicosanoids, biological oxidative metabolites of arachidonic acid (AA), play essential roles in cellular homeostasis as well as in therapeutic applications [9, 10]. These lipid mediators, which include prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETEs), are generated from cyclooxygenases (COXs), lipoxygenases (LOXs), and http://dx.doi.org/10.3343/alm.2016.36.4.342 Lee SH, et al. LC-MS-based in vitro eicosanoid metabolic assay CYP ω-hydroxylase and through non-enzymatic processes. Compared with the analysis of individual circulating inflammation markers such as nuclear factor κB, interleukin (IL)-6, IL-10, C-reactive protein, and tumor necrosis factor-α (TNF-α) [2, 8], the metabolite profiling of signaling mediators may reveal the biological consequences of inflammation. Gas chromatography-mass spectrometry (GC-MS)-based profiling of eicosanoids is generally performed with chemical derivatization because of their low volatility and polar nature [11, 12]. However, the lack of analytical stability and assay accessibility limits its use for clinical applications, and instead liquid chromatography-mass spectrometry (LC-MS) is widely used [6, 8, 13-15]. In particular, LC-MS-based lipidomic platforms are useful for both qualitative and quantitative determination of eicosanoids in biological specimens [8, 13-15]. Given the complex regulation of eicosanoid metabolism, it is beneficial to simultaneously quantify various types of lipid mediators rather than to quantify only a limited number of eicosanoids. Therefore, a method for LC-MS-based quantitative profiling of 32 eicosanoids was developed and validated using rabbit liver S9 fractions. The devised in vitro assay was then used to quantify the metabolites of AA catalyzed by COXs, LOXs, and CYP ω-hydroxylase in liver S9 fractions obtained from rabbits with systemic inflammation, which was induced by injecting 1% carrageenan (CGN) subcutaneously. In addition to corticosteroids, which act as potent anti-inflammatory agents in the treatment of chronic inflammation [16, 17], endogenous androgens play an important role in regulating inflammation and immune responses [18, 19]. After confirming the validity of our assay using two known COX inhibitors, celecoxib for COX-2 and ibuprofen for COX-1/2, an in vitro metabolic assay was applied to investigate the new biochemical functions of the androgen epitestosterone, which has complex and diverse metabolic functions in endocrine physiology [20, 21]. METHODS 1. Materials Reference standards of the 32 eicosanoids used in this study (Table 1) were obtained from Cayman Chemical (Ann Arbor, MI, USA). The following internal standards were also purchased from Cayman Chemical: 3,3,4,4-d 4-6-keto PGF1α for 6-keto PGF1α, 3,3,4,4-d 4-PGE2 for the seven prostaglandins and for 11-dehydro TXB2, 3,3,4,4-d 4-8-iso PGF2α for the ten isoprostanes, 6,7,14,15-d 4-LTB4 for the four leukotrienes, 5,6,8,9,11, 12,14,15-d 8-12(S)-HETE for TXB2 and for the five hydroxyeicohttp://dx.doi.org/10.3343/alm.2016.36.4.342 saeonic acids, and 5,6,8,9,11,12,14,15-d 8-AA for AA. Epitestosterone was obtained from Steraloids (Newport, RI, USA). For solid-phase extraction (SPE), an Oasis MAX cartridge (1 mL, 30 mg; Waters; Milford, MA, USA) was preconditioned with 1 mL methanol followed by 1 mL deionized water. L-Ascorbic acid (reagent-grade) and for (...truncated)