Structural characterization of monohydroxyeicosatetraenoic acids and dihydroxy- and trihydroxyeicosatrienoic acids by ESI-FTICR

Lijie Cui 0 1 3 Marilyn A. Isbell 0 1 3 Yuttana Chawengsub 0 1 3 John R. Falck 0 1 2 William B. Campbell 0 1 3 Kasem Nithipatikom 0 1 3 0 Address reprint requests to Dr. Kasem Nithipatikom, Department of Pharmacology and Toxicology, Medical College of Wisconsin , 8701 Water- town Plank Road, Milwaukee, Wisconsin 53226, USA 1 Published online January 31, 2008 Received August 21, 2007 Revised January 17, 2008 Accepted January 17, 2008 2 Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center , Dallas, Texas, USA 3 Department of Pharmacology and Toxicology, Medical College of Wisconsin , Milwaukee, Wisconsin, USA The fragmentation characteristics of monohydroxyeicosatetraenoic acids and dihydroxy- and trihydroxyeicosatrienoic acids were investigated by electrospray ionization Fourier transform ion cyclotron resonance (FTICR) mass spectrometry using sustained off-resonance irradiation collision-induced dissociation (SORI-CID) and infrared multiphoton dissociation (IRMPD). The fragmentation patterns of these compounds were associated with the number and positions of the hydroxyl substituents. The fragmentation is more complicated with increasing number of the hydroxyl groups of the compounds. In general, the major carbon- carbon cleavage of [M H] ions occurred at the -position to the hydroxyl group, and the carbon- carbon cleavage occurred when there was a double-bond at the -position to the hydroxyl group. SORI-CID and IRMPD produced some common fragmentation patterns; however, each technique provided some unique patterns that are useful for structural identification of these compounds. This study demonstrated the application of FTICR via the identification of regioisomers of trihydroxyeicosatrienoic acids in rabbit aorta samples. (J Am Soc Mass Spectrom 2008, 19, 569 -585) 2008 American Society for Mass Spectrometry - strates the utility of SORI-CID and IRMPD for the structural characterization of monohydroxyeicosatetraenoic acids (HETEs) and dihydroxy- and trihydroxyeicosatrienoic acids (DHETs and THETAs). HETEs, DHETs, and THETAs are metabolites of arachidonic acid (AA). Different isoforms of lipoxygenases (LOX) metabolize AA to regioisomeric HETEs. 12-Hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and 15-HETE are important lipid mediators in inflammation, kidney, immune system, prostate diseases, and diabetes [714]. Cytochrome P450 epoxygenases metabolize AA to 4 regioisomeric epoxyeicosatrienoic acids (EETs), and soluble epoxide hydrolase (sEH) enzymes subsequently convert EETs to the corresponding DHETs [1517]. EETs have various biological functions, including inhibiting the hydro-osmotic action of arginine vasopressin in the kidney, calcium mobilization, and prostaglandin formation [18, 19]. EETs stimulate relaxation in coronary rings and coronary arterioles [20 25]. A recent study showed that 14,15-DHET is a potent peroxisome proliferator-activated receptor(PPAR ) activator in COS-7 cells [26]. THETAs are new members of the family of endothelium-derived relaxing factors [2731]. 11,12,15THETA and 11,14,15-THETA were identified as endothelium-derived lipoxygenase metabolites of AA in Mtant tool for analysis of biomolecules. Fourier ass spectrometry has emerged as an importransform ion cyclotron resonance (FTICR) mass spectrometry is one of the techniques that can provide high mass accuracy and high mass resolution [1 6]. Accurate molecular weight, elemental composition, and structural information can be achieved from FTICR. Sustained off-resonance irradiation collisioninduced dissociation (SORI-CID) and infrared multiphoton dissociation (IRMPD) are two different dissociation techniques often used with FTICR for MS/MS analysis. SORI-CID involves collision of the target ion slightly off its resonance frequency with a collision gas, causing the acceleration and deceleration of ions during the RF pulse. At a frequency of several kilohertz, multiple low-energy collisions occur as ions are vibrationally excited for a sustained period. Unlike SORICID, no collision gas is required for IRMPD. Instead, the 75 W CO2 laser is used to irradiate the ions to form fragments. The fragments may continue to acquire some energy from the infrared laser pulse and further fragment to ions of lower masses. This study demonthe rabbit aorta. Recent studies have shown that THETAs relax rabbit small mesenteric arteries [31]. 11,12,15THETA mediates acetylcholine-induced relaxations by activating apamin-sensitive potassium (K ) channels in vascular smooth muscle to induce K efflux, membrane hyperpolarization, and vascular relaxation [27, 28], while 11,14,15-THETA is not vasoactive [28, 29]. These studies cogently indicate that biological function is dependent upon the position of the hydroxyl groups. Previous studies showed that HETEs and DHETs form characteristic fragments during MS2 analysis by ion trap and triple quadrupole mass spectrometers [32 40]. The mechanisms for electrospray ionization and tandem mass spectrometry of various classes of eicosanoids have been elegantly reviewed [41]. In this study, we investigated the mass spectrometric characteristics of HETEs, DHETs, and THETAs by ESI-FTICR using SORI-CID and IRMPD. The effects of the number and the positions of the hydroxyl substituents on fragmentation patterns were characterized, and the identities of THETAs in biological samples were determined. Materials and Methods 11-, 12-, and 15-HETE; 11,12- and 14,15-DHET; 14,15EET, and arachidonoyl dopamine were purchased from Cayman Chemical Co. (Ann Arbor, MI). 11,12,15-, 11,14,15-, and 13,14,15-THETA were synthesized in the laboratory of Dr. J. R. Falck [42]. Indomethacin, A23187, and L-ascorbic acid were purchased from Sigma (St. Louis, MO). C18 Bond Elut solid-phase extraction (SPE) columns were purchased from Varian (Harbor City, CA). Acetonitrile was HPLC grade. Distilled, deionized water was used in all experiments. Biological Sample Preparation Tissue preparation and incubation. Aortas were isolated from 1- to 2-week old New Zealand White rabbits (Kuiper Rabbit Ranch, Gary, IN), placed in ice-cold HEPES buffer (in mM; 10 HEPES, 150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, and 6 glucose; pH 7.4), cleaned of adhering connective tissue and fat, and cut into rings (5-mm long). Aortic rings were incubated for 10 min at 37 C in HEPES buffer containing indomethacin (10 5 M). AA (10 4 M) was added, and the vessels were incubated for an additional 5 min. Calcium ionophore A23187 (2 10 5 M) was added, and the vessels were incubated for another 15 min. The reaction was stopped by the addition of ethanol to a final concentration of 15%. The incubation buffer was removed, acidified (pH 3.5) with glacial acetic acid, and extracted on Bond Elut C-18 extraction columns as previously described [2729]. The extracts were evaporated to dryness under a stream of N2 and stored at 40 C until further HPLC separation. Separation of AA metabolites by HPLC. The extra (...truncated)