Identification of glutathione adducts of α-chlorofatty aldehydes produced in activated neutrophils.

α-Chlorofatty aldehydes (α-ClFALDs) are produced by hypochlorous acid targeting plasmalogens during neutrophil activation. This study investigated the reaction of the α-chlorinated carbon of α-ClFALD with the nucleophile, GSH. Utilizing ESI/MS/MS, the reaction product of GSH and the 16-carbon α-ClFALD, 2-chlorohexadecanal (2-ClHDA), was characterized. The resulting conjugate of 2-ClHDA and GSH (HDA-GSH) has an intact free aldehyde, and the chlorine at the α-carbon is ejected. Stable isotope-labeled [d4]HDA-GSH was synthesized, which further confirmed the structure, and was used to quantify natural α-ClFALD conjugates of GSH (FALD-GSH) using reverse-phase LC with detection by ESI/MS/MS using selected reaction monitoring. HDA-GSH is elevated in RAW 264.7 cells treated with physiologically relevant concentrations of exogenous 2-ClHDA. Furthermore, PMA-treated primary human neutrophils have elevated levels of HDA-GSH and the conjugate of 2-chlorooctadecanal (2-ClODA) and GSH (ODA-GSH), as well as elevated levels of 2-ClHDA and 2-ClODA. Production of both conjugates in PMA-stimulated neutrophils was reduced by 3-aminotriazole pretreatment, which also blocks endogenous α-ClFALD production. Additionally, plasma FALD-GSH levels were elevated in the K/BxN mouse arthritis model. Taken together, these studies demonstrate novel peptidoaldehydes derived from GSH and α-ClFALD in activated human neutrophils and in vivo in K/BxN mice.


ESI/MS/MS characterization of 2-ClHDA and GSH in vitro reaction products
TLC-purifi ed 2-ClHDA conjugates of GSH (HDA-GSH) was diluted 2,000× with acetonitrile-water (7:3) containing 0.1% formic acid, which was analyzed by ESI/MS/MS by direct infusion at a fl ow rate of 5 l/min. For ESI/MS/MS, the ionization energy and temperature were set at 3,700 V and 270°C for positive ion mode and 2,600 V and 270°C for negative ion mode. A collision energy of 15 eV and collision gas of 1.0 Torr argon were used for MS/MS analyses in both the positive and negative ion modes.

DNPH derivatization of HDA-GSH
Sixty micrograms of synthetic HDA-GSH was suspended in 100 l of water-acetonitrile (1:1, v/v) in a reaction vessel. One hundred microliters of 3.1 mg/ml DNPH in 2 M HCl was added, and the reaction was vortexed every minute for 15 min. Subsequently, 1 ml of water was added, and the reaction product was purifi ed using a C-18 solid-phase extraction (SPE ) column (Supelclean™ LC-18 SPE Tube, 100 mg). Reaction products were eluted with 1 ml methanol. The eluted product was dried under N 2 and suspended in 300 l water-acetonitrile (3:1, v/v). Free DNPH was then removed with three washes of 500 l chloroform. The remaining aqueous layer was dried and suspended in 200 l wateracetonitrile-isopropanol (3:3:2, v/v/v) containing 0.15% formic acid. Ten microliters of the SPE-purifi ed derivative was diluted 50-fold and analyzed by ESI/MS/MS in direct injection mode.

Synthesis of stable isotope-labeled HDA-GSH
Commercially available [7,7,8,8-d 4 ]hexadecanoic acid was converted to 2-bromo- [7,7,8,8-d 4 ]hexadecanal  ]HDA) as previously described ( 24 ). One hundred and fi fty milligrams of 2-Br-[ d 4 ]HDA was then reacted with 300 mg GSH in 3 ml of ethyl ether-methanol-water (2:1.5:0.6, v/v/v) for 4 h at 37°C. Reaction products were then extracted in ethyl ether by the addition of 10 ml water and 15 ml ethyl ether. The collected ethyl ether layer including interface was extracted and dried to a white solid. The white solid was sequentially washed three times with 10 ml hexane, two times with 10 ml methanol, and two times with 10 ml water to remove any remaining free 2-Br-[ d 4 ]HDA and GSH. The fi nal product was dried by lyophilization, weighed, and the purity of the resultant [ d 4 ]HDA-GSH product confi rmed by direction infusion ESI/MS/MS.

RAW 264.7 cell treatments with 2-ClHDA
RAW 264.7 cells were treated with either 0, 1, 5, 25, or 50 M 2-ClHDA in DMEM supplemented with 2% FBS for 8 h. The medium was then removed, and cells were washed with PBS. The cells were scraped in 1.25 ml PBS containing 10 mM NEM and immediately frozen. NEM was added to the fi nal cell homogenate solution to block free GSH sulfhydryls from spontaneously reacting with ␣ -ClFALD following cell incubations.

Human neutrophil studies
Whole blood (50-150 ml) was taken from healthy volunteers and anticoagulated with EDTA (fi nal concentration 5.4 mM) prior to the isolation of neutrophils using a Ficoll-Hypaque gradient as previously described ( 9 ). These studies were approved and authorized by the Saint Louis University Institutional Review Board Protocol 9952. Informed consent was obtained from the human subjects. Neutrophils were diluted to 1 million neutrophils per ml in HBSS. PMA (200 nM) in ethanol (0.1%) and 10 mM 3-aminotriazole (AT) were then added as indicated. Incubations forms reversible Schiff base adducts with amines ( 20 ), the electrophilic properties of the ␣ -chlorinated carbon of ␣ -ClFALD have not been examined as a target of cellular nucleophiles. The metabolism of lipid oxidation products such as 4-hydroxy-2-( E )-nonenal ( 21 ), biosynthesis and metabolism of leukotrienes ( 22 ), and detoxifi cation of xenobiotics ( 23 ) are all heavily reliant on conjugation to glutathione (GSH). Because the nucleophilic thiol of GSH is known to modify unsaturated and halogenated carbons either chemically or catalyzed by glutathione-S -transferase ( 23 ), the studies herein examined the electrophilic nature of the ␣ -chlorinated carbon of ␣ -ClFALD as a target of the nucleophilic thiol of glutathione.
Understanding ␣ -ClFALD metabolism and the cellular impact of ␣ -ClFALD metabolites is critical to better understand the role of chlorinated lipids in infl ammatory pathologies. Accordingly, the present study examined the reactivity of 2-chlorohexadecanal (2-ClHDA) with GSH and the production of ␣ -ClFALD conjugates of GSH (FALD-GSH) in activated neutrophils. The results demonstrate that ␣ -ClFALD chemically reacts with GSH to produce a novel class of peptidoaldehydes (FALD-GSH) and identify FALD-GSH production in activated neutrophils as well as in vivo in the plasma of a mouse arthritis model.

Instrumentation
MS was performed using a Thermo Fisher TSQ Quantum Ultra mass spectrometer (Thermo Fisher, Waltham, MA). For experiments requiring LC/MS, a Thermo Fisher Surveyor LC system was coupled to the Quantum Ultra. LC/MS data analysis was performed using XCalibur software (Thermo Fisher).

2-ClHDA and GSH in vitro reaction products and TLC purifi cation
Unless otherwise indicated, all reactions were carried out in PBS (pH 7.4), methanol, and ethyl ether at a ratio of 0.6:1.5:2. Briefl y, 2-ClHDA or hexadecanal (HDA) (3 mol) was fi rst suspended in 73 l of ethyl ether in a clean, borosilicate reac tion vessel. Fifty-fi ve microliters of methanol was then added ± N -ethylmaleimide (NEM; 2.25 mol). Finally, GSH (1.5 mol) was added in 22 l PBS, and the reaction mixture was vortexed and allowed to react in a sealed reaction vessel at 37°C for 4 h. Reaction aliquots (10 l) were loaded onto 40 Å silica gel TLC plates. Mobile phase for TLC was composed of chloroform-acetonemethanol-water-acetic acid (6:8:2:2:1 v/v/v/v/v). TLC plates where visualized with ninhydrin or 2,4-dinitrophenylhydrazine (DNPH). TLC lanes used for purifi cation were not stained but scraped and extracted using 1 ml of acetonitrile-water (7:3, v/v) supplemented with 0.25% formic acid. The solution was centrifuged at 2,000 g for 5 min and the supernatant removed. The supernatant was dried under nitrogen and suspended in 150 l screened by fl uorescence-activated cell sorting analysis as previously described ( 27 ). CD4 V ␤ 6-positive mice were maintained on normal chow and euthanized at 9 weeks to obtain whole blood via cardiac puncture. Mice at this age have signifi cant joint swelling of all four paws, and 50 l of serum from these mice can robustly induce arthritis in C57BL/6J mice ( 27 ). To measure FALD-GSH, 45 fmol of [ d 4 ]HDA-GSH was added to 25 l of plasma and sequentially diluted with 150 l water containing 0.1% formic acid and then 640 l methanol-acetonitrile (3:1, v/v). Diluted plasma was subsequently centrifuged 10 min at 16,100 g max at 4 ° C to pellet precipitated protein. The resultant supernatant was collected and diluted with 470 l water containing 0.1% formic acid prior to purifi cation and quantifi cation of FALD-GSH using Strata-X columns and LC/MS/MS (vide supra). Total plasma ␣ -ClFA molecular species were measured from 25 l of plasma subjected to base hydrolysis in the presence of 105 fmol 2-chloro-[ d 4 ]hexadecanoic acid (internal standard), and total fatty acid was extracted ( 11,26 ). Fatty acids were then subjected to reverse-phase HPLC using a Phenomenex Onyx monolithic C-18 column (50 × 2.0 mm) and detected using ESI/MS/MS by SRM as previously described ( 11,26 ).

Statistical analyses
Student's t -test was used for comparisons between two groups. For comparisons between three or more groups to the control condition ANOVA was performed with the Dunnett post hoc test. In other cases when appropriate, Tukey's post hoc test was used. All data are presented as mean ± SEM unless otherwise indicated.

In vitro adducts of 2-ClHDA with GSH
Based on the premise that the ␣ -carbon of ␣ -ClFALD is a target for modifi cation by cellular nucleophiles, we examined the reactivity of 2-ClHDA with GSH ( Fig. 1A ). In initial studies, 2-ClHDA and GSH were reacted at a 2:1 molar excess of 2-ClHDA for 4 h. Reaction products were separated on silica gel TLC, and a novel reaction product was detected (R f ≈ 2.4) using either ninhydrin or DNPH staining ( Fig. 1B, C , lane 3). Both the reaction product and GSH are stained by ninhydrin ( Fig. 1B ), which readily stains the amine of GSH but does not stain 2-ClHDA. Additionally, the reaction product is stained by DNPH ( Fig.  1C ), which stains aldehydes but does not stain GSH. The importance of the ␣ -chlorinated carbon for GSH attack is highlighted by the demonstration that no novel products were for 0-60 min at 37°C . At the end of each reaction, 10 mM NEM was added, and the neutrophils were immediately frozen prior to extraction.

Extraction and quantifi cation of FALD-GSH
To extract and quantitate FALD-GSH, 90 fmol of [ d 4 ]HDA-GSH and 1 vol of methanol-acetonitrile (1:1, v/v) were added to 100 l of the RAW 264.7 cell homogenate, and 45 fmol of [ d 4 ]HDA-GSH and 1 vol of methanol-acetonitrile (1:1, v/v) were added to 1 ml neutrophil suspension. Both solutions were then vortexed for 30 s, sonicated in a sonication bath for 15 s, and vortexed for another 30 s. The resulting solution was centrifuged at 1,000 g for 5 min, and the supernatant removed. The supernatant was loaded on a Strata-X column (60 mg bed weight) that was preconditioned with 1.2 ml methanol followed by 1.2 ml water-methanol (4:1, v/v). Columns were washed two times with 0.6 ml water-methanol (4:1, v/v), and then HDA-GSH was eluted with 1.2 ml of methanol-acetonitrile (3:1, v/v) containing 0.25% formic acid. The eluted adduct was dried under nitrogen and suspended in 100 l of 6:4:5 acetonitrile-isopropanol-water containing 0.15% formic acid for analysis by LC/MS/MS. LC was performed using a Phenomenex Onyx C-18 column (50 × 2.0 mm) with ESI/MS/MS detection using selected reaction monitoring (SRM). Two mobile phases were used for LC. Solvent A was water containing 0.15% formic acid, and solvent B was acetonitrile-isopropanol (3:2, v/v) containing 0.15% formic acid. Initial column conditions were 65/35 (A/B) at a fl ow rate of 200 l/min, which was held for 2 min following the injection of 25 l of the Strata-X purifi ed solutions onto the column. Subsequently, GSH adducts of 2-ClHDA were eluted by a 3 min linear gradient to 100% solvent B.  formation. Taken together, nucleophilic attack of GSH on the ␣ -chlorinated carbon, TLC evidence that the product contains amines and a free aldehyde, and the positive ion spectra shown in Fig. 2B support the structure shown in Fig. 2D . This proposed product has a C-S bond bridging the GSH cysteine to the ␣ -carbon of 2-ClHDA while retaining a free aldehyde. This fatty aldehyde-GSH (FALD-GSH) adduct molecular species, HDA-GSH has a molecular mass of 545.31. Additional minor ions found in the positive ion scan of HDA-GSH are at m/z 568.32 and 528.30, which represent the sodiated adduct and the protonated adduct with the loss of water, respectively. The fourth ion found in the positive survey scan, m/z 399.30, is a common fragment of GSH adducts of lipids ( 28 ), which corresponds to the loss of glutamate and water from the parent ion, m/z 546.36. The TLC-purifi ed HDA-GSH adduct was also subjected to negative ion ESI/MS, which complements the positive ion scan. The m/z 399.41 fragment ion corresponds with the loss of were observed in reactions between GSH and HDA, which does not contain an ␣ -chlorinated carbon ( Fig. 1 B, C , lane 5). In other reactions, NEM was added to the reaction solution just prior to GSH. Under these conditions, 2-ClHDA did not form an adduct with GSH ( Fig. 1B, C , lane 6), indicating that NEM is a preferred target for GSH compared with 2-ClHDA, and GSH adducts with 2-ClHDA are through a thiol-related mechanism. The NEM-GSH reaction product was visualized with the ninhydrin stain ( Fig. 1B , lane 6).

Characterization of the reaction product of 2-ClHDA and GSH by MS
To characterize the product of the reaction of GSH with 2-ClHDA, the novel band (R f ≈ 2.4) resolved on TLC was extracted and then subjected to ESI/MS/MS. Initially, the TLCpurifi ed reaction product was subjected to a second TLC step to confi rm the purity of the material as well as confi rm the stability of the product after purifi cation ( Fig. 2A ). In positive ion mode using ESI/MS, the purifi ed reaction product was detected as a major ion at m/z 546.36 ( Fig. 2B ). This spectra indicates the product is not monochlorinated because there is no corresponding signature M + 2 ion associated with 37 Cl. Thus, nucleophilic attack of the ␣ -chlorinated carbon of ␣ -ClFALD results in chlorine ejection during adduct , fragments into multiple ions ( Fig. 3B ) that directly correspond to the known fragmentation of GSH ( 29,30 ). The predominating ion, m/z 272.16, is the cleavage of HDA-GSH at the sulfhydryl as shown in the Fig. 3B inset. Both the positive and negative ion survey scan and parent ion fragmentations support the structure proposed in Fig. 2D .

Characterization of DNPH-HDA-GSH
TLC-purifi ed HDA-GSH as shown in Fig. 1C was readily stained by DNPH as HDA-GSH retains a free aldehyde. Based on this fi nding, the purifi ed DNPH derivative of HDA-GSH was analyzed by ESI/MS/MS. In positive survey mode ( Fig. 4A ), the DNPH-HDA-GSH derivative is detected as the [M + H] + at m/z 726.19, which is consistent with DNPH forming a hydrazone with HDA-GSH with a molecular mass of 725.34 ( Fig. 4C ). MS/MS of m/z 726.19 yielded fragment positive ions that were consistent with that observed with fragmentation of HDA-GSH (e.g., m/z 399.39 and 296.32) as well as new ions from GSH fragmentation ( m/z 308.21, 179.05, and 162.05).

HDA-GSH quantitation in chemical reactions and HDA-GSH production in RAW 264.7 cells treated with 2-ClHDA
Using the LC/MS/MS method for the quantifi cation of FALD-GSH, we tested the time-dependent incorporation of 20 M 2-ClHDA into FALD-GSH adducts in the presence of 2 mM GSH. Under these conditions ‫ف‬ 6% of the 2-ClHDA precursor is converted to FALD-GSH per hour, and this rate is linear for up to 4 h ( Fig. 7A ). To determine maintaining a constant [ d 4 ]HDA-GSH level. The response plot is shown in Fig. 6B , which demonstrates that [ d 4 ] HDA-GSH and HDA-GSH give an equal ion intensity response with a calibration line with a slope = 1. Furthermore, the calibration response was similar between natural HDA-GSH and deuterated HDA-GSH in the presence of neutrophil lysate extract in the solvent matrix ( Fig. 6C ). This detection method provides reliable detection of HDA-GSH at levels as low as 5 fmol.  and then analyzed for HDA-GSH production. RAW 264.7 cells were chosen as they are a well-characterized macrophage cell line, but incapable of producing 2-ClHDA. To enhance sensitivity and reproducibility of detection, HDA-GSH was fi rst purifi ed from cell homogenate using Strata-X columns as described in the Materials and Methods. No HDA-GSH was detected in the control treatment (ethanol vehicle alone was added), while HDA-GSH levels were elevated in a precursor-dependent manner between 1 and 50 nmol 2-ClHDA ( Fig. 7B ).

HDA-GSH and ODA-GSH accumulation in PMAstimulated neutrophils
Isolated primary human neutrophils stimulated with PMA produce ␣ -ClFALD ( 9 ) and were, therefore, assayed for the production of ␣ -ClFALD adducts of GSH.   and ODA-GSH production on ␣ -ClFALD and MPO activity in activated neutrophils.

DISCUSSION
␣ -ClFALD production in MPO-containing phagocytes and during infl ammatory processes has previously been described (9)(10)(11)(12). However, the mechanisms underlying the biological properties and metabolism of ␣ -ClFALD are 2-ClODA molecular species were elevated by 5 min, peak by 30 min, and begin to decline by 60 min. Interestingly, the production of HDA-GSH and ODA-GSH adducts do not directly mirror 2-ClHDA and 2-ClODA levels at these time points ( Fig. 8B ). Instead, there appears to be differential conversion of 2-ClHDA and 2-ClODA to their GSH adducts. Importantly, both 2-ClHDA and 2-ClODA levels were reduced in 200 nM PMA-treated neutrophils at 30 min that were pretreated with an inhibitor (AT) that prevents HOCl production ( Fig. 9A ). Likewise, HDA-GSH and ODA-GSH levels were signifi cantly reduced in ATpretreated, PMA-treated neutrophils ( Fig. 9B ). Inhibition of ␣ -ClFALD and their GSH adducts (HDA-GSH and ODA-GSH) by AT demonstrate the dependence of HDA-GSH  signature ion in the adduct product (e.g., M and M + 2 ions at a 3 to 1 ratio due to the natural abundance of 35 Cl and 37 Cl were not observed). MS/MS analyses of HDA-GSH and [ d 4 ]HDA-GSH revealed the common loss of glutamate and water yielding the robust fragment ions m/z 399 and m/z 403, respectively, These fragment ions were used for both SRM detection of HDA-GSH and [ d 4 ]HDA-GSH and method development to quantify FALD-GSH species in biological samples sequentially subjected to SPE and LC/MS/MS. Response calibration lines comparing ion response of HDA-GSH and [ d 4 ] HDA-GSH in the presence and absence of biological matrix (extracted neutrophil lysate) had slopes of 1 demonstrating equal ion intensity response of the natural and deuterated HDA-GSH. It is also likely, and assumed, that ODA-GSH will have a similar response compared with [ d 4 ]HDA-GSH because the ionization of the precursor and product is dependent on the protonated amine groups of the peptide component of the parent ion and product ion. Utilizing this method, the production of HDA-GSH in RAW 264.7 mouse macrophage cells treated with 2-ClHDA was examined. Under these conditions, HDA-GSH increased in a concentration-dependent manner in the presence of physiological amounts of 2-ClHDA ( 9, 10 ). RAW 264.7 cells produce very minimal MPO activity without prolonged stimulation ( 32 ), and therefore, it is unlikely that endogenous 2-ClHDA contributed to HDA-GSH production. The LC/ MS/MS method was fi nally used to show that human neutrophils stimulated with PMA produce HDA-GSH and ODA-GSH from endogenously produced ␣ -ClFALD. The dependence of HDA-GSH and ODA-GSH production on MPO activity in activated neutrophils was confi rmed by the ability of the heme enzyme inhibitor, AT ( 33 ), to ablate the accumulation of both ␣ -ClFALD and FALD-GSH molecular species in PMA-stimulated neutrophils.
Previous studies have examined the metabolism of ␣ -ClFALD produced in PMA-stimulated neutrophils to ␣ -ClFA and ␣ -ClFOH ( 17,18 ). In those studies, both ␣ -ClFA and ␣ -ClFOH were elevated 15 min following PMA stimulation, although ␣ -ClFALD was elevated as early as 5 min. Similar to ␣ -ClFA and ␣ -ClFOH production, HDA-GSH and ODA-GSH are elevated at 15 min, which collectively suggest that ␣ -ClFALD is cleared by either oxidation ( ␣ -ClFA), reduction ( ␣ -ClFOH), or nucleophilic attack by GSH. Interestingly, relative levels of HDA-GSH and ODA-GSH did not mirror relative levels of 2-ClHDA and 2-ClODA throughout 60 min of PMA stimulation. HDA-GSH levels were signifi cantly higher than ODA-GSH at 15 min, HDA-GSH and ODA-GSH levels were similar at 30 min, and ODA-GSH was elevated over HDA-GSH at 60 min. In comparison with this changing relative amount of the FALD-GSH species, the relative amounts of 2-ClHDA and 2-ClODA remain the same throughout stimulation with more 2-ClODA present at all time points examined. These fi ndings might be explained by GSH having disparate accessibility to 2-ClHDA and 2-ClODA over time following PMA stimulation. Although GSH can chemically target ␣ -ClFALD in neutrophils, it is also possible that adducts are formed by glutathione S -transferase activity ( 21,22 ), and it is possible not completely known. The present study reveals for the fi rst time the production of a novel class of peptidoaldehydes resulting from ␣ -ClFALD being targeted by GSH nucleophilic attack. We predicted that the ␣ -chlorinated carbon of ␣ -ClFALD would be reactive with the GSH sulfhydryl group. Indeed, a unique reaction product was found as a result of treating ␣ -ClFALD with GSH. The attack on this fatty aldehyde was dependent on the presence of the ␣ -chlorinated carbon because nonchlorinated HDA did not react with GSH. Furthermore, the reaction product from the treatment of 2-ClHDA with GSH retained a free aldehyde as well as amine groups as demonstrated by DPNH and ninhydrin staining of TLC-purifi ed reaction products, respectively ( Fig. 1 ). These fi ndings also supported the structure of the adduct shown in Fig. 2D , which would be produced from GSH attack of the ␣ -chlorinated carbon accompanied by chlorine ejection. This structure was further supported by the following: 1 ) positive and negative ion MS/MS of the 2-ClHDA-GSH adduct product, 2 ) MS/MS in the positive ion mode of the DNPH hydrazone derivative of the 2-ClHDA-GSH adduct product, 3 ) analogous survey mode and MS/MS mode molecular ions with a 4 amu shift of the 2-ClHDA-GSH adduct containing 4 deuteriums, and 4 ) the absence of a chlorine the role of this lipid as a lipid mediator is not fully understood due to its metabolism and chemical reactivity. Results herein provide new insights into the fates and potential mediators of ␣ -ClFALD produced as a result of MPO activity by demonstrating that ␣ -ClFALD is targeted by the nucleophile GSH. Several functions have been attributed to ␣ -ClFALD, and it now seems possible that some of these biological functions might be attributed to peptidoaldehyde FALD-GSH production in target cells.
that either glutathione S -transferase or GSH used by glutathione S -transferase has disparate accessibility or molecular species preference in forming FALD-GSH molecular species over the time course of PMA stimulation (e.g., 60 min). It should also be noted that neutrophil GSH levels change over the time course of PMA stimulation due to the robust production of oxidants during neutrophil stimulation. It has been reported in human neutrophils that GSH levels decrease from 1.3 to 0.65 nmol per million neutrophils in response to 80 min of PMA stimulation ( 34 ). GSH is a target for HOCl during neutrophil activation ( 34,35 ), and this may limit FALD-GSH formation in activated neutrophils. However, it should be appreciated that even with GSH reduced due to neutrophil activation, GSH levels exceed that of the maximal levels of ␣ -ClFALD (e.g., ‫ف‬ 0.1 nmol ␣ -ClFALD per million neutrophils following 30 min of PMA stimulation). It will be interesting in future studies to examine adduct formation from neutrophil-produced ␣ -ClFALD by neighboring cells such as endothelial cells that are spared, or have minimal exposure to, neutrophil-derived HOCl. Additionally, in vivo studies with the K/BxN mouse show elevated plasma levels of both FALD-GSH and ␣ -ClFA compared with C57Bl/6J mice. At the time (9 weeks) that plasma was collected, K/BxN mice display a previously described arthritic phenotype with joint swelling in all four paws, and one of the major mediators of this injury is through neutrophil activation ( 27,31 ). These data are the fi rst to show elevated chlorinated lipids derived from plasmalogen oxidation as well as elevations in the newly discovered FALD-GSH adducts in this arthritis model.
There are similarities in structure between FALD-GSH and leukotriene C4 (LTC4), with each containing GSH coupled to a lipid oxidation product. It is possible that FALD-GSH is metabolized by pathways similar to those previously characterized for cysteinyl leukotrienes including LTC4 transport out of cells, extracellular removal of the glutamate residue by ␥ -glutamyl transferase to yield leukotriene D4, dipeptidase activity resulting in leukotriene E4 (LTE4) production, and N -acetylation of LTE4 ( 22 ). Data herein show the FALD-GSH level produced by activated neutrophils is ‫ف‬ 250 fmol per million cells, and it is possible that this level is higher in either neighboring cells that are exposed to ␣ -ClFALD or in neutrophils at longer postactivation times. In comparison, LTC4 levels in A23187-activated neutrophils ranges from ‫ف‬ 50 to 700 fmol per million cells ( 36,37 ). Taken together, FALD-GSH and LTC4 levels are likely produced at sites of neutrophil activation at levels within an order of magnitude of each other. In addition to identifying potential parallel metabolic pathways of FALD-GSH and peptidoleukotrienes, it will also be important in future studies to examine the biological properties of FALD-GSH and the potential interactions FALD-GSH may have with peptidoleukotrienes.