Activation of Human Polymorphonuclear Leukocytes by Products

Oxidation of red blood cell (RBC) ghost preparations initiated by tert-butyl hydroperoxide (tBuOOH) was employed to explore the formation of lipid pro...
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Chem. Res. Toxicol. 1998, 11, 1024-1031

Activation of Human Polymorphonuclear Leukocytes by Products Derived from the Peroxidation of Human Red Blood Cell Membranes Lisa M. Hall and Robert C. Murphy* Department of Pediatrics, Division of Basic Sciences, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206 Received May 22, 1998

Oxidation of red blood cell (RBC) ghost preparations initiated by tert-butyl hydroperoxide (tBuOOH) was employed to explore the formation of lipid products derived from endogenous phospholipids that specifically expressed biological activity toward the human polymorphonuclear leukocyte (PMN). Common measure of lipid peroxidation, thiobarbituric acid-reactive substances (TBARS) and the increased absorbance at 235 nm consistent with the formation of conjugated dienes, was observed following a 90-min incubation of RBC ghosts with tBuOOH. Saponification of phospholipids and separation of the resultant fatty acids by RP-HPLC permitted direct mass spectrometric analysis of oxidized fatty acids. Individual HPLC fractions were assayed for their ability to increase intracellular free calcium ion concentrations in human PMN to guide structural investigations. Two fractions were found to contain biologically active components, and tandem mass spectrometric analysis of the abundant ions observed in these fractions resulted in the characterization of several oxidized polyunsaturated fatty acids derived from arachidonic and linoleic acids. The major components in these fractions included 5-hydroxyeicosatetraenoic acid (5-HETE) and 5-hydroperoxyeicosatetraenoic acid (5-HpETE). The dose-dependent increases in intracellular calcium in the neutrophil using synthetic 5(rac)HETE, 5(rac)-HpETE, and 5-oxo-ETE were found to have EC50’s of 250, 6, and 3 nM, respectively. The quantity of 5-oxygenated arachidonate components present in oxidized RBC was consistent with the observed biological response elicited by fractions A and B. This study suggests that 5-HETE and 5-HpETE are abundant products of lipid peroxidation of cellular membranes and that these racemic products possess significant biological activity. Such compounds could play important roles as mediators of the cellular response to toxicologic stimuli that generate free radical species.

Introduction Enzymatic oxidation of arachidonic acid by prostaglandin H synthase, lipoxygenases, and cytochrome P450 results in the production of biologically active oxidized products including prostaglandins, thromboxanes, leukotrienes, and hydroxyeicosatetraenoic acids such as 5-, 12-, and 15-HETE,1 and epoxyeicosatetraenoic acids (13). These eicosanoids are known to play important roles as lipid mediators of various physiologic and pathophysiologic responses (4). As the molecular mechanisms responsible for the initial oxidation of free arachidonic acid catalyzed by these iron-containing enzymes are becoming elucidated, it is clear that these enzymes carefully control the formation and reactivity of bisallylic carbon-centered radicals with molecular oxygen. Formation of such delocalized radical intermediates of arachi* Corresponding author. Tel: (303) 398-1849. Fax: (303) 398-1694. E-mail: [email protected]. 1 Abbreviations for oxidized polyunsaturated fatty acids employed in this manuscript follow the nomenclature proposal [Smith et al. (1990) Methods Enzymol. 187, 1-9]: 5-HETE, 5-hydroxy-6,8,11,14eicosatetraenoic acid; 5-HpETE, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid; 5-oxo-ETE, 5-keto-6,8,11,14-eicosatetraenoic acid; 12HETE, 12-hydroxy-5,8,10,14-eicosatetraenoic acid; 15-HETE, 15hydroxy-5,8,11,13-eicosatetraenoic acid; 9-HODE, 9-hydroxy-10,12octadecadienoic acid; 9-HpODE, 9-hydroperoxy-10,12-octadecadienoic acid; 9-oxo-ODE, 9-keto-10,12-octadecadienoic acid.

donic acid can also result from free radical reactions not under enzymatic control (5). The discovery of compounds isomeric to the prostaglandins including F2-isoprostanes (6), isothromboxanes (7), and E2-isoprostanes (8) has now been reported in several relevant systems. Free radical oxidation of arachidonate has also been found to generate compounds isomeric to leukotrienes, termed B4-isoleukotrienes (9). An important feature of the production of compounds isomeric to enzymatically derived eicosanoids is that the free radical events take place while the arachidonic acid is esterified to glycerophospholipids within cellular membranes, whereas cyclooxygenase, 5-lipoxygenase, and cytochrome P450 utilize free arachidonic acid as substrate (1-3, 6, 9). Of further interest is that several of these free radical-derived eicosanoids have been found to possess significant biological activity including F2-isoprostanes (10) and B4-isoleukotrienes (9). These observations have focused attention on such nonenzymatic derived products of arachidonic acid as markers of lipid peroxidation and also as compounds that may mediate toxicological events when reactive oxygen species are generated within tissues or cells. Within the normal cell, multiple mechanisms exist to prevent propagation of free radical reactions by trapping highly reactive radicals with concomitant formation of stabilized and long-lived radical species. Reduced glu-

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Active Peroxidation Products from Red Blood Cells

tathione plays a central role in terminating radical reaction through glutathione peroxidase and formation of oxidized glutathione (11). Significant energy is expended in the maintenance of reduced glutathione levels to protect cells from damage initiated by formation of a radical species or exposure to radicals from the extracellular milieu. The red blood cell has been widely studied for its resistance to lipid peroxidation through various defense mechanisms (12-14). However, under pathophysiologic conditions where chronic exposure to radical-generating systems can occur, these defense mechanisms may be overwhelmed. For example, during the inflammatory response involving the human polymorphonuclear leukocyte, high concentrations of reactive oxygen species are generated as cytotoxic agents in regions where cells such as red blood cells may coexist. Oxidized lipid products are likely formed under such conditions and, if released, could initiate signal transduction events within adjacent cells. A detailed characterization of the most abundant biologically active products generated during lipid peroxidation of naturally occurring membranes and their ability to activate human neutrophils has not been previously reported. The present study was undertaken to investigate lipid peroxidation in the red blood cell (RBC) ghost which had been depleted of glutathione and defense mechanisms to limit lipid peroxidation. The biological activity of the resulting mixture of compounds was assessed for their ability to increase intracellular calcium ion in human neutrophils. With this bioassay as a guide, the structures of the most abundant biologically active products were determined largely using techniques of mass spectrometry.

Methods Materials. Resveratrol, arachidonic acid-d8, 5(S)-HETE-d8, 5(S)-HETE, racemic 5-HETE, 5(S)-HPETE, racemic 5-HPETE, 5-oxo-ETE, 13(S)- and 9(S)-HpODE, 9- and 13-oxo-ODE, and LTB4 were obtained from Cayman Chemical (Ann Arbor, MI). The following chemicals were obtained from Sigma Chemical Co. (St. Louis, MO): tBuOOH, butylated hydroxytoluene (BHT), L-ascorbic acid, malonaldehyde bis(dimethyl acetal) (MDA), and 2-thiobarbituric acid (TBA). INDO-1/AM was obtained from Calbiochem (La Jolla, CA). All solvents were HPLC grade (Fisher, Fair Lawn, NJ). Preparation of RBC Ghosts. Venous blood (30 mL) was taken from normal human volunteers, treated with 3.8% sodium citrate (3.3 mL), and within 60 min centrifuged at 300g for 20 min to separate serum and buffy coat from RBC. The cells were washed by resuspending them in 50 mL of phosphate-buffered saline (PBS; 8.0 g of NaCl, 0.20 g of KCl, 1.51 g of Na2HPO4, 1.16 g of KH2PO4 dissolved in 1 L of water and adjusted to pH 7.4) and then centrifuged at 760g for 15 min to pellet the cells. A second wash in 50 mL of PBS was employed followed by a final centrifugation at 1700g for 15 min to yield packed RBCs. Red blood cell lysis and RBC membranes (ghosts) were prepared as previously described (15). Incubation with tBuOOH. RBC ghosts (2 mL, 0.2 mg of protein/mL) were incubated in the presence and absence (control incubation) of 10 mM tBuOOH for 90-min at 37 °C. In separate experiments, RBC ghost preparations were preincubated with 1 mM BHT, 5 mM ascorbic acid, or 0.44 mM resveratrol for 1 min prior to the addition of tBuOOH followed by a 90 min incubation at 37 °C. Each reaction was terminated by the addition of 5 mg of BHT dissolved in 1 mL of methanol to limit any further oxidation during lipid extraction. Thiobarbituric Acid-Reactive Substances (TBARS) Assay. Analysis of the production of TBARS was performed

Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1025 essentially by the method of Moore and Brummitt (16). Briefly, an aliquot of the incubation media (400 µL) was removed and added to 0.85 mL of thiobarbituric acid (0.47%) also containing 0.4 g of trichloroacetic acid. This solution was then boiled for 15 min and allowed to cool before measurement of UV absorption at 532 nm. Standard curves in the range of 0-0.4 nmol/ mL were prepared according to the method of Jain (17). Lipid Extraction and Saponification. Phospholipids were extracted from RBC ghosts essentially by the method of Bligh and Dyer (18) substituting methylene chloride for chloroform. The methylene chloride layer was removed, taken to dryness, and then resuspended in 1.5 mL of 85% methanol. Hydrolysis of the fatty acyl groups was carried out at room temperature for 1 h by the addition of 500 µL of NaOH (1 N). The reaction mixture was acidified by the addition of 50 µL of 88% formic acid and the sample vacuum-concentrated to approximately 200 µL. The sample was then diluted to 1 mL with methanol/ acetonitrile (35:65). The stable isotope-labeled internal standard 5(S)-HETE-d8 (25 ng) was added at this point to those samples taken for quantitative analysis of 5-HETE as previously described (19). Gas Chromatography/Mass Spectrometry (GC/MS) Analysis of Fatty Acids. An aliquot (1%) of the saponified fatty acids obtained from RBC ghosts treated with tBuOOH, as well as RBC ghosts that were not treated, was analyzed by negative ion chemical ionization GC/MS to detect the major fatty acids present from RBC membranes. Internal standard (arachidonic acid-d8, 10 ng) was added to samples as well as to a standard curve (0-50 ng) prepared for docosahexaenoic and arachidonic acids. Samples and standard curves were extracted with hexane, and the aqueous phase was removed. After drying, pentafluorobenzyl esters (PFB) were prepared as previously described (20). Reverse-Phase HPLC/Mass Spectrometry (RP-HPLC/ MS). An Ultremex 5-µm, 4.6-mm × 250-mm column (Phenomenex, Torrance, CA) with an ODS Guard-pak precolumn (Waters, Marlborough, MA) was used for separation of saponified fatty acyl groups. The solvent system was as follows: solvent A, 8.3 mM acetic acid adjusted to pH 5.7 with ammonium hydroxide; solvent B, methanol/acetonitrile (35:65). The flow rate was 1 mL/min with the following stepwise gradient: 3055% B over the first 10 min, 55-75% B over the next 15 min, 75-100% B over 5 min, then hold at 100% B. The effluent was split postcolumn with 30% diverted to the mass spectrometer. Intracellular Calcium Assay. Human neutrophils were prepared from the whole blood of healthy volunteers and treated with INDO-1/AM as previously described (21). An aliquot (50%) of each collected HPLC fraction was taken to dryness under vacuum and then dissolved in 100 µL of Hank’s balanced salt solution (HBSS). Neutrophil response and desensitization were assessed before addition of each HPLC fraction (10-50 µL) by the response to LTB4 (2 nM). Calculations for the conversion of fluorescence signal into intracellular calcium concentrations were performed according to Tsien et al. (22). Dose-response curves were analyzed by nonlinear regression using convergence on a best-fit curve to a sigmoidal dose-response equation. Electrospray Ionization Multiple Reaction Monitoring (MRM) Mass Spectrometry. Mass spectrometry analysis was performed on a Sciex API-III+ instrument (PE-Sciex, Toronto, Canada) operated in the negative ion mode with an orifice voltage of -55 V. Curtain gas flow was 1.2 L/min, nebulizer pressure was 40 psi, and ion spray voltage was -3500 V (nitrogen as nebulizing gas). Product ion spectra were obtained using a collision energy of 15 eV and collision gas thickness (argon) of 211 × 1012 molecules/cm2.

Results Lipid Peroxidation. Incubation of red blood cell (RBC) ghosts for 90 min with tBuOOH (10 mM) resulted in a significant increase in TBARS (1.69 ( 0.36 nmol/ mg of protein) compared to that observed in untreated

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Table 1. Production of TBARS Following Incubation of RBC Ghosts with TBuOOH (10 mM) for Various Times time (min)

TBARSa (nmol/mg of protein)

0 30 60 90 120

0.31 ( 0.12 1.15 ( 0.20 1.39 ( 0.68 0.77 ( 0.19 0.78 ( 0.14

a Samples are averages ( SEM of three separate incubations of RBC ghosts derived from the same blood donor.

RBC ghosts (0.667 ( 0.05 nmol/mg of protein) from three separate subjects, similar to that previously reported (23-25). The increase in TBARS was found to maximize at 60 min (Table 1). The mechanism responsible for reduction in TBARS at later times was not further investigated. Furthermore, the formation of TBARS was inhibited by the antioxidants BHT (0.1 nM, 75% inhibition) and resveratrol (0.04 mM, 55.4% inhibition). Ascorbic acid (10 mM), however, was found to increase 2.2fold the formation of TBARS in RBC ghosts treated with tBuOOH. This may be expected due to the ability of ascorbic acid to maintain ferrous iron that catalyzes further decomposition of hydroperoxides by the Fenton reaction (26). While this increase in TBARS and further inhibition by antioxidants suggested the formation of free radical lipid peroxidation products in this model, the exact chemical nature of the oxidized fatty acyl group could not be ascertained from this assay alone because of the variety of precursor structural modalities that would yield malonyldialdehyde-like products during the assay conditions. Detailed structural studies of the fatty acyl peroxidation products were obtained following extraction of phospholipids, saponification, and reverse-phase HPLC to separate the oxidized and nonoxidized fatty acids from control and tBuOOH-treated RBC ghosts (Figure 1). A large number of new fatty acid components were present in treated ghosts as indicated by the ultraviolet absorption at 235 nm for the effluent between 25 and 30 min (Figure 1A) which was not observed in untreated RBC ghosts (Figure 1B). These HPLC retention times were found to be precisely those observed for the elution of various synthetic, oxidized polyunsaturated fatty acids, including 5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acids (HETE) as well as corresponding hydroperoxyeicosatetraenoic acids (HpETE), oxidized octadecadienoic acids including 9- and 13-hydroxyoctadecadienoic acids (HODE) and 9- and 13-hydroperoxyoctadecadienoic acids (HpODE). Each of these oxidized fatty acids contains a conjugated diene structural unit and thereby displays a UV absorption maximum at 235 nm. Additional components of the tBuOOH-treated RBC ghosts eluted in this same HPLC retention time region but had significant UV absorption at 280 nm (inset, Figure 1A). This region was found to be the retention time expected for oxidized compounds such as 9- and 13-oxooctadecadienoic acid (oxo-ODE) and 5-, 12-, and 15-oxoeicosatetraenoic acid (oxo-ETE). Such components were also absent in the control incubation. The abundant constituents in both the control and treated RBCs eluting between 34 and 38 min (Figure 1) were identified by electrospray mass spectrometry and gas chromatography/mass spectrometry of the corresponding collected HPLC fractions. The majority of the compounds in these fractions corresponded to polyun-

Figure 1. RP-HPLC separation of fatty acyl species from RBC ghosts. Phospholipids were extracted from RBC ghosts and saponified to release esterified fatty acids. Separation of oxidized and nonoxidized fatty acids was performed by RP-HPLC with UV detection at 235 nm from (A) RBC ghosts treated with tBuOOH (10 mM; 90 min at 37 °C) and (B) control RBC ghosts which were incubated for 90 min at 37 °C without the addition of tBuOOH. Inset represents absorbance at 280 nm. Fractions (1 min) were collected for biological assay and further mass spectrometric investigation.

saturated fatty acids including arachidonic acid and linolenic acid which eluted between 34 and 35 min and linoleic acid between 35 and 36 min. These naturally occurring fatty acids were observed eluting from the HPLC at 235 nm due to the end absorption of UV light from their nonconjugated double bonds and the large quantity of these polyunsaturated fatty acids present in the extract. Biological Activity of HPLC Fractions. Oneminute RP-HPLC fractions from the separation of saponified RBCs phospholipids isolated from tBuOOHtreated RBC were collected and tested for induction of an increase in intracellular calcium ion in INDO-1/AMloaded human neutrophils. Three fractions, A, B, and C corresponding to fractions collected at 27-28, 28-29, and 29-30 min, respectively, were found to substantially increase neutrophil intracellular calcium (Figure 2). As discussed previously, these fractions were expected to contain both oxidized arachidonic and linoleic acid species. HPLC fractions prior to A and B and after 30 min did not elicit a significant increase in neutrophil intracellular calcium. Furthermore, corresponding HPLC fractions collected during separation of saponified fatty acyl species from control RBC ghosts (not incubated with tBuOOH) did not elicit an increase in intracellular calcium ion. Immediately after the addition of HPLC fractions to the neutrophil assay and the return of the fluorescence

Active Peroxidation Products from Red Blood Cells

Figure 2. Calcium response of RP-HPLC fractions. Fluorescence signal indicative of changes in the intracellular free calcium ion concentration elicited from several RP-HPLC fractions eluting between 23 and 33 min (Figure 1) from RBC ghosts treated with tBuOOH. Fractions prior to A, B, and C, as well as those after, were not active. Fractions A (corresponding to HPLC fraction 28, collection between 27 and 28 min), B (corresponding to HPLC fraction 29, collection between 28 and 29 min), and C (corresponding to HPLC fraction 30, collection between 29 and 30 min) elicited a significant increase in intracellular calcium concentrations in human neutrophils. LTB4 (6 pmol) was added directly after the return of the signal to baseline to assess the degree of desensitization of the LTB4 receptor due to the active compounds present.

signal to baseline, 6 pmol of LTB4 was added to ascertain if elevation of intracellular calcium would again be elicited or if desensitization of the LTB4 receptor had occurred (27). LTB4 caused a significant increase in intracellular calcium suggesting that the compounds present in fractions A, B, and C were not exerting their effects primarily through the LTB4 receptor (Figure 2). Although fraction C initially responded in this neutrophil assay, subsequent purification, including normal phase HPLC, resulted in the loss of all biological activity, and no attempts were made to further pursue identification of active components in this fraction. It is likely that the active component in this fraction degraded during sample handling. Structural Characterization of Oxidized Fatty Acids. Summation of the mass spectral data from fractions A (Figure 3A) and B (Figure 3B) obtained during online LC/MS of treated RBC ghosts revealed carboxylate anions indicative of the presence of numerous oxidized fatty acid components, as anticipated. The most abundant components had negative ions (carboxylate anions) between m/z 290 and 350 when background ions as well as ions present in those corresponding HPLC fractions from untreated RBC ghosts were subtracted. Carboxylate anions present in fraction A (Figure 3A) were consistent with the elution of several expected oxidized linoleic and arachidonic acid species including 9- and 13-oxo-ODE (m/z 293), 9- and 13-HODE (m/z 295), 9- and 13-HpODE (m/z 311), various oxo-ETEs (m/z 317), various HETEs (m/z 319), and various HpETEs (m/z 335). The carboxylate anion at m/z 393 was not further characterized but did maximize in this region. Carboxylate anions found in fraction B (Figure 3B) were consistent with the elution of various species including: 9- and 13-oxo-ODE (m/z 293), 9- and 13-HODE (m/z 295), various oxo-ETEs (m/z 317), various HpETEs (m/z 335), and oxidized (keto) 22:4 (m/z 345). Components generating negative ions at m/z 393, 395, and 419 were not

Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1027

Figure 3. Reconstructed electrospray (negative ion) mass spectrum of HPLC fractions containing components active in elevating neutrophil intracellular calcium ion. Summation of mass spectral scans (LC/MS) recorded at 27-28 min (A) and 28-29 min (B) during elution of fatty acids derived from RBC ghosts. Oxygenated arachidonic acid species yield carboxylate anions at m/z 319, 317, and 335. Oxidized species of linoleic acid yield ions at m/z 293, 295, and 311. These species were not detected in control RBC ghosts. Nonoxidized fatty acids palmitate (16:0), stearate (18:0), and docosahexanoate (22:6) yield ions at m/z 255, 283, and 327, respectively.

further identified; however, m/z 297 corresponded to a hydroxyoctadecanoate, but the position of the double bond and the hydroxyl group was not further investigated. Several other fatty acids were observed in these fractions but were also present in the untreated RBC ghost extract including fatty acids 16:0 (m/z 255), 18:0 (m/z 283), and 22:6 (m/z 327). Oxidized products of arachidonic and linoleic acid were not detected in the mass spectral data corresponding to the regions described above from control RBC ghosts. The major oxidized arachidonic and linoleic acid species in each fraction represented by these carboxylate anions were further analyzed by collision-induced decomposition (CID) and tandem mass spectrometry of fractions A and B, and their identifications are listed in Table 2. Previous mass spectrometric studies of the decomposition of the carboxylate anion from various unsaturated hydroxy fatty acids revealed specific product ions characteristic of the position of double bonds as well as the nature of the oxygen substitution (28). Tandem mass spectrometry of m/z 293 yielded abundant product ions at m/z 113 and 125 consistent with 13-oxo-ODE and 9-oxo-ODE, respectively. The ion at m/z 293 could also have been derived from the corresponding 13-hydroperoxyoctadecadienoic acid (13-HpODE) and 9-hydroperoxyoctadecadienoic acid (9-HpODE) which have a common carboxylate anion at m/z 311 and undergo rapid dehydration to yield m/z 293 (29). Collision-induced decomposition of m/z 319 (Figure 4A) present in fraction A representing elution from the HPLC between 27 and 28 min resulted in abundant ions at m/z 203 and 115 which likely arose from cleavage of the

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Table 2. Tandem Mass Spectrometric Analysis (MS/MS) and Identification of Carboxylate Anions [M - H]Derived from Fatty Acid Hydrolysis Products of RBC Ghost Phospholipids following Treatment with tBuOOH (10 mM) HPLC fraction (retention product ionsa time, min) [M - H]- CID [M-H]- structural assignmentb 25-26

319 295

26-27

319 335/317c 311/293 295

27-28 min (A)

319 335/317 311/293 295

28-29 min (B)

335/317 311/293 295

219 171 195 208, 179 167 139, 151, 167 113 113 125 171 195 167 163, 155 115, 203 153 113 125 171

15-HETE 9-HODE 13-HODE 12-HETE 11-HETE 9-HETE 15-HpETE/15-oxo-ETE 13-HpODE/13-oxo-ODE 9-HpODE/9-oxo-ODE 9-HODE 13-HODE 11 -HETE 8-HETE 5-HETE 12-HpETE/12-oxo-ETE 13-HpODE/13-oxo-ODE 9-HpODE/9-oxo-ODE 9-HODE

195 203/129 113 125 171 195

13-HODE 5-HpETE/5-oxo-ETE 13-HpODE/13-oxo-ODE 9-HpODE/9-oxo-ODE 9-HODE 13-HODE

a Abundant product ion(s) following collision-induced dissociation (CID) of the corresponding carboxylate anion [M - H]- using electrospray ionization and a tandem quadrupole mass spectrometer. b Assignment of structure based upon the previously reported production spectra derived from the corresponding carboxylate anion [M - H]-. c Electrospray ionization of lipid hydroperoxides results in a facile loss of the elements of water. These dehydration products are mass spectrometrically indistinguishable from the corresponding oxo compound.

carbon-carbon bond adjacent to a hydroxyl group and a double bond which would be consistent with CID events taking place at carbon 6 of 5-HETE. This CID spectrum was remarkably similar to that obtained from authentic 5-HETE (Figure 4B) as previously described (28). The ions at m/z 301 corresponded to the loss of water from the carboxylate anion and that at m/z 257 from loss of water and carbon dioxide. The ion at m/z 167 was not present in the collision-induced decomposition mass spectrum of authentic 5-HETE but was a major ion in the collision-induced decomposition of the carboxylate anion of 11-HETE; however, the abundance of this isomer maximized in the previous HPLC fraction (Table 2). Another isomer, 8-hydroxyeicosatetraenoic acid (8HETE), was also present in this fraction as revealed by product ions at m/z 163 and 155 derived from m/z 319 (Figure 4A). Other hydroxyeicosatetraenoic acid isomers were found to elute in earlier HPLC fractions (Table 2), including 15-hydroxyeicosatetraenoic acid (15-HETE) (between 25 and 26 min) which yielded an abundant product ion at m/z 219 from collision-induced decomposition of m/z 319 and 9-, 11-, and 12-hydroxyeicosatetraenoic acid (12-HETE) which yielded abundant ions from m/z 319 at m/z 139/151/167, 167, and 208/179, respectively, which were found in the previous HPLC fraction representing 26-27 min. The CID fragmentation ions for the family of HETEs have been previously described (28) and the fragmentations determined to occur based on the position of the double bonds and the hydroxyl moiety.

Figure 4. CID tandem mass spectrometry of m/z 319 and 317 derived from major oxidized arachidonic acid species. CID tandem mass spectrometry was performed on the major oxidized arachidonic species present in biologically active fractions A and B, corresponding to collection between 27 and 28 min and 2829 min, from RBC ghosts treated with tBuOOH. (A) CID of m/z 319 from fraction A and with several HETE species corresponding to 5-, 8-, and 11-HETE is presented. However, the most abundant species was 5-HETE. The structure of 5-HETE is shown with the characteristic fragmentation sites annotated. (B) CID of m/z 317 derived from facile dehydration of facile species during electrospray ionization as indicated. The most abundant HpETE/oxo-ETE species in fraction B was 5-HpETE/ oxo-ETE. Ions characteristic for 12- and 15-HpETE/oxo-ETE were not observed in fraction B.

Collision-induced decomposition of the ion at m/z 317 from fraction A was found to yield a characteristic ion at m/z 153 that would be derived from 12-oxoeicosatetraenoic acid (12-oxo-ETE). The presence of 12-HpETE in this fraction could not be ruled out as also being present since its carboxylate anion at m/z 335 would also readily decompose to m/z 317 and 153. It was clear that this fraction collected between 27 and 28 min was a mixture of several oxidized fatty acid substances derived from octadecadienoic acid as well as arachidonic acid. The components present in fraction B were also analyzed by tandem mass spectrometry in a similar manner. The most abundant arachidonate-containing component was represented by the carboxylate anion at m/z 317. Collision-induced decomposition of this ion (Figure 4B) revealed a product ion mass spectrum virtually identical to that for 5-oxoeicosatetraenoic acid (5-

Active Peroxidation Products from Red Blood Cells

Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1029 Table 3. Determination of 5-HETE, 5-HpETE, and 5-Oxo-ETE Formed following Treatment of RBC Ghosts with tBuOOH (10 mM) at Various Incubation Timesa nmol/ng of protein time (min) 0 30 90

5-HETE ndb