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Originally published In Press as doi:10.1074/jbc.M602505200 on April 11, 2006

J. Biol. Chem., Vol. 281, Issue 25, 16849-16860, June 23, 2006
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Metabolism of Myeloperoxidase-derived 2-Chlorohexadecanal*

Kristin R. Wildsmith, Carolyn J. Albert, Dhanalakshmi S. Anbukumar, and David A. Ford1

From the Department of Biochemistry and Molecular Biology, St. Louis University Health Sciences Center, St. Louis, Missouri 63104

Received for publication, March 16, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Numerous studies have suggested relationships between myeloperoxidase (MPO), inflammation, and atherosclerosis. MPO-derived reactive chlorinating species attack membrane plasmalogens releasing {alpha}-chloro fatty aldehydes including 2-chlorohexadecanal (2-ClHDA), which have been found to accumulate in activated neutrophils, activated monocytes, infarcted myocardium and human atheromas. The present study employed synthetically prepared 2-Cl-[3H]-HDA as well as stable isotope-labeled 2-ClHDA to elucidate the metabolism of 2-ClHDA. The results herein demonstrate that human coronary artery endothelial cells oxidize and reduce 2-ClHDA to its respective chlorinated fatty acid ({alpha}-ClFA) and chlorinated fatty alcohol ({alpha}-ClFOH). Within the first hour of incubations of human coronary artery endothelial cells with 2-Cl-[3H]-HDA, the label was incorporated into the {alpha}-ClFOH and {alpha}-ClFA pools. After 1 h, the radiolabel was predominantly found in the {alpha}-ClFOH pool. Cell-derived {alpha}-ClFOH and {alpha}-ClFA were also released into the cell culture medium. Additionally, chlorinated fatty acid was incorporated into complex endothelial cell glycerolipids, including monoglycerides, triglycerides, phosphatidylcholine, and phosphatidylethanolamine. The oxidation and reduction of 2-ClHDA to {alpha}-ClFA and {alpha}-ClFOH, respectively, was further supported by mass spectrometric analyses of human coronary artery endothelial cells incubated with either 2-ClHDA or stable isotope-labeled 2-ClHDA (2-Cl-[d4]-HDA). 2-ClHDA was also oxidized to {alpha}-ClFA and reduced to {alpha}-ClFOH in both control and phorbol 12-myristate 13-acetate-stimulated neutrophils. Taken together, these results show that a family of chlorinated lipidic metabolites is produced from {alpha}-chloro fatty aldehydes derived from reactive chlorinating species targeting of plasmalogens. These metabolites are incorporated into complex lipids and their biological roles may provide new insights into MPO-mediated disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phagocytes are important mediators of host defense mechanisms against microbes (1–3). The concomitant release of myeloperoxidase (MPO)2 with its substrate, H2O2, by phagocytes results in the production of the reactive chlorinating species (RCS), hypochlorous acid (HOCl), which is a bactericidal agent (4). The production of HOCl by MPO amplifies the oxidizing potential of reactive oxygen species produced by the phagocyte respiratory burst (2). HOCl elicits its antimicrobial actions and cytotoxicity by targeting multiple critical molecules including proteins, nucleic acids, and lipids (5–13). For example, HOCl reacts with primary amine-containing molecules, resulting in the production of the more stable RCS, monochloramines and dichloramines (14). Tyrosine residues of proteins can also be targeted by RCS resulting in 3-chlorotyrosine (15, 16).

In addition to the antimicrobial actions of MPO-derived HOCl, it also targets host tissues and likely participates in the pathophysiological sequelae of several cardiovascular diseases, including atherosclerosis and myocardial ischemia-reperfusion injury (17–22). Recently, RCS have been shown to target the plasmalogen molecular subclass of phospholipids, resulting in the unmasking of the plasmalogenic vinyl ether aliphatic group as an {alpha}-chloro fatty aldehyde (23). This halogenated aldehyde accumulates in both human atherosclerotic lesions and infarcted rat myocardium (17, 18). Furthermore, at physiological concentrations, {alpha}-chloro fatty aldehyde is a chemoattractant and has been shown to elicit cardiac injury and ventricular dysfunction (18, 24). The accumulation of {alpha}-chloro fatty aldehyde in atherosclerotic lesions and in infarcted myocardium, coupled with the potential role this lipid may have on cardiovascular function, underscore the importance of determining the mechanisms responsible for {alpha}-chloro fatty aldehyde catabolism.

Many aldehydes found in vivo are produced through free radical mechanisms. The cytotoxicity of aldehydes is attenuated by reduction, oxidation, or conjugation with glutathione (25–30). Aldehyde dehydrogenase oxidizes the aldehyde to its carboxylic acid. Human liver microsomal aldehyde dehydrogenase is specific for medium- and long-chain fatty aldehydes (31). A mutation in this fatty aldehyde dehydrogenase has been linked to Sjögren-Larsson syndrome (32). Alternatively, long-chain fatty aldehydes can be reduced to their respective fatty alcohols (33–35). Additionally, aldehydes can diffuse within, or escape from, their cells of origin and form covalent adducts with proteins and phospholipids. Reactive aldehydes have been shown to react with primary amines on proteins to form Schiff base adducts and Michael addition products (formed by the nucleophilic attack on an {alpha},beta-unsaturated aldehyde such as 4-hydroxynonenal). In fact, 4-hydroxynonenal has been shown to covalently modify low density lipoprotein apolipoproteins contributing to the formation of a high uptake form of low density lipoprotein and to form protein adducts during ischemia-reperfusion injury (36, 37). Another aldehyde, p-hydroxyphenylacetaldehyde, a product of MPO oxidation of L-tyrosine, forms Schiff base adducts with aminophospholipids of low density lipoprotein (38), which are present in human atherosclerotic tissues (39).

Understanding the metabolism of {alpha}-chloro fatty aldehyde may be critical in revealing the role of this novel plasmalogen oxidation product in cardiovascular disease. Accordingly, the present study was designed to elucidate the metabolism of the {alpha}-chloro fatty aldehyde, 2-chlorohexadecanal (2-ClHDA). The results herein demonstrate that both endothelial cells and neutrophils oxidize and reduce 2-ClHDA to its respective chlorinated fatty acid and chlorinated fatty alcohol, which are subsequently incorporated into complex glycerolipids.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Synthesis and Purification of 2-Cl-[d4]-HDA and 2-Cl-[3H]-HDA—2-Cl-[d4]-HDA was synthesized and purified as previously described (24). 2-Cl-[3H]-HDA was synthesized and purified by a modification of the synthetic scheme employed for 2-Cl-[d4]-HDA (24). For this scheme, [9,10-3H]-hexadecanol was prepared by the reduction of [9,10-3H]-hexadecanoic acid (0.256 Ci/mmol, PerkinElmer Life Sciences). The purity of the synthetic 2-Cl-[3H]-HDA was determined by TLC of underivatized 2-Cl-[3H]-HDA and TLC of its corresponding dimethylacetal and pentafluorobenzyl oxime derivatives using petroleum ether/ethyl ether/acetic acid (90/10/1, v/v/v) as the mobile phase.

Synthesis of 2-Chlorohexadecanoic Acid and 2-Chlorohexadecanol—Hexadecanoic acid (16:0 fatty acid (FA)) and [d4]-16:0 FA were subjected to {alpha}-chlorination with chlorine(g) using the Hell-Volhard-Zelinsky reaction and phosphorous as the catalyst (40). Briefly, 16:0 FA was melted at 80 °C before an equimolar amount of phosphorous trichloride in dichloromethane was added to the reaction vial. Chlorine(g) was then slowly bubbled into the reaction mixture for 1 h. The crude product was sequentially extracted using a modified Bligh and Dyer method (41), TLC-purified (silica gel G TLC plates with a mobile phase composed of petroleum ether/ethyl ether/acetic acid (70/30/1, v/v/v) (RF = 0.24)), and then further purified on a Thermo Finnigan Surveyor liquid chromatograph equipped with a Beckman RP UltrasphereTM ODS (5 µ, 4.6 mm x 25 cm) column coupled to a TSQ Quantum Ultra triple quadrupole mass spectrometer. Products were eluted at a flow rate of 2 ml/min from the stationary phase with a mobile phase composed of 85/15 MeOH/H2O containing 0.02% formic acid (A) for 3 min followed by a linear gradient from 100% A to 100% MeOH (B) over 7 min. The solid phase was further eluted with 100% B for 10 min. The LC eluate was split 10:1, and the LC-purified reaction products were detected by electrospray ionization-mass spectrometry using selected ion monitoring (SIM) of m/z 289 ([M – H]– of 2-Cl-16:0 FA) or m/z 293 ([M – H]– of [d4]2-Cl-16:0 FA) and selected reaction monitoring (SRM) for the loss of HCl of m/z 289 -> m/z 253 or m/z 293 -> m/z 257 in the negative ion mode (electrospray needle voltage = 5 kV, capillary T = 320 °C, collision energy = 15 eV) (tr for the 2-Cl-16:0 FA and [d4]2-Cl-16:0 FA were 8.55 and 8.53 min, respectively).

2-ClHDA and 2-Cl-[d4]-HDA were resuspended in 2 ml of radical free ethyl ether and 0.5 ml of benzene and treated with VitrideTM reagent (sodium bis(2-methoxyethoxy)aluminum hydride) for 30 min at 37 °C (42). The resultant 2-chlorohexadecanol ({alpha}-ClFOH) was purified by TLC (petroleum ether/ethyl ether/acetic acid (70/30/1, v/v/v)) (RF = 0.41).

2-ClHDA Metabolism in Human Coronary Artery Endothelial Cells—Human coronary artery endothelial cells (HCAEC) (Cell Applications, Inc.) were grown to confluency on 60-mm tissue culture plates (Corning) in EGM®-2-MV medium (Cambrex) supplemented with 5% fetal bovine serum following the supplier's instructions at 37 °C with 100% humidity and 5% CO2 (passages 4–10). Confluent HCAEC were incubated with selected concentrations of 2-Cl-[3H]-HDA (0.1 µM (256 mCi/mmol), 1 µM (256 mCi/mmol), or 10 µM (25.6 mCi/mmol)) in 5 ml of normal growth medium at 37 °C for selected time intervals. At the end of each experimental interval, lipids in the cell culture medium were extracted by the method of Bligh and Dyer (41). The cell culture plates containing the cells were then washed with phosphate-buffered saline. Subsequently, the cells were scraped with methanol:water (1:1, v/v) prior to lipid extraction by the method of Bligh and Dyer (41). Lipids extracted from the cells or culture medium lipids were stored in chloroform under nitrogen prior to analyses by chromatography.

For pulse-chase experiments, HCAEC were incubated (pulse) with 1 µM 2-Cl-[3H]-HDA (256 mCi/mmol) for 30 min. Following this 30-min pulse radiolabeling interval, the cell culture medium was removed from HCAEC, and the cells were washed once with phosphate-buffered saline and then incubated with cell culture medium containing 10 µM 2-ClHDA for selected time intervals (chase). At the end of each chase interval, radiolabeled lipid metabolites of 2-Cl-[3H]-HDA associated with the HCAEC, as well as the cell culture medium, were prepared for analyses (see above).

Parallel experiments were performed using stable isotope-labeled 2-ClHDA. In these experiments, HCAEC were incubated with 10 µM 2-Cl-[d4]-HDA for either 3 or 24 h. Extracting the lipids from the cells and cell culture medium terminated the incubations, as described above. Stable isotope-labeled metabolites of 2-ClHDA were subsequently purified by TLC and analyzed by electrospray ionization-mass spectrometry or GC-MS.

2-Cl-[3H]-HDA Metabolism in Human Neutrophils—Whole blood (50 ml) was taken from healthy volunteers and anti-coagulated with EDTA (final concentration 5.4 mM) prior to the isolation of neutrophils using a Ficoll-Hypaque gradient as previously described (43). Pelleted neutrophils (5 x 106 cells/condition) were resuspended in Hanks' balanced salt solution (pH 7.3) supplemented with both MgSO4 and CaCl2 at 1 mM and immediately incubated with 1 µM 2-Cl-[3H]-HDA (256 mCi/mmol) in the presence or absence of 200 nM phorbol 12-myristate 13-acetate for 0, 15, 30, or 60 min at 37 °C. The cells were pelleted and washed once with Hanks' balanced salt solution before the lipids were extracted by the method of Bligh and Dyer (41).

Thin Layer Chromatographic Analyses of 2-ClHDA Metabolites—Neutral lipid metabolites of 2-Cl-[3H]-HDA were purified from crude lipid extracts using silica gel 60 TLC plates (Whatman) as a solid phase with a mobile phase composed of petroleum ether/ethyl ether/acetic acid (70/30/1, v/v/v) (44). Alternatively, polar lipid metabolites of 2-Cl-[3H]-HDA were purified on the same solid phase but with a mobile phase composed of chloroform/methanol/water (65/35/4, v/v/v). Lipid metabolites were further characterized by two-dimensional TLC plates using silica gel 60 TLC plates and an initial mobile phase composed of chloroform/methanol/ammonia (65/25/5, v/v/v) followed by development in the second dimension with a mobile phase composed of chloroform/acetone/methanol/acetic acid/water (3/4/1/1/0.5, v/v/v/v/v). Radioactivity associated with lipid metabolites of 2-Cl-[3H]-HDA that was resolved on TLC plates was detected by fluorography. In brief, the developed TLC plates were treated with EN3HANCE spray (PerkinElmer Life Sciences) prior to exposure to x-ray film (Kodak) at –80 °C. Alternatively, parallel TLC plates or lanes (on TLC plates) were developed with lipid standards, and silica from regions associated with specific lipids was scraped from the plate. Radioactivity was quantified by liquid scintillation spectrometry. Additionally, silica associated with regions of TLC plates detected by fluorography was also scraped from the plate and quantified by liquid scintillation spectrometry.

Chlorinated Fatty Alcohols—TLC-purified chlorinated fatty alcohols were converted to their respective pentafluorobenzoyl esters using 2,3,4,5,6-pentafluorobenzoyl chloride (PFB-Cl) at 60 °C for 45 min (45). GC-MS analysis of PFB esters was performed using a Hewlett Packard (Palo Alto, CA) 6890 gas chromatograph and 5973 mass spectrometer using the negative ion chemical ionization mode with methane as the reagent gas. The source temperature was set at 150 °C. The electron energy was 240 eV, and the emission current was 300 mA. The PFB derivatives were separated on a J & W Scientific (Folsom, CA) DB-1 column (12.5 m, 0.2 mm inner diameter, 0.33 mm film thickness). The injector and the transfer line temperatures were maintained at 250 °C. The GC oven was maintained at 150 °C for 3.5 min, increased at a rate of 30 °C/min to 270 °C, and held at 270 °C for an additional 2 min.


Figure 1
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  		FIGURE 1.
TLC of synthetic 2-Cl-[3H]-HDA. A, synthetic 2-Cl-[3H]-HDA and 2-ClHDA were subjected to TLC and detection as described under "Experimental Procedures." Additional lanes as indicated contain the dimethylacetal (DMA) and pentafluorobenzyl (PFB) oxime derivatives of synthetic 2-Cl-[3H]-HDA and 2-ClHDA. 2-Cl-[3H]-HDA and its derivatives were not detected by charring but were visualized by autoradiography (lanes 2, 4, and 6). 2-ClHDA and its derivatives were visualized by charring (lanes 1, 3, and 5). Images of the charring and autoradiography were overlaid. B, 2-Cl-[3H]-HDA was subjected to TLC and fluorography to enhance the detection of potential minor contaminants.

 


Figure 2
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  		FIGURE 2.
Two-dimensional TLC analyses of 2-ClHDA metabolites in HCAEC. HCAEC were incubated with 1 µM 2-Cl-[3H]-HDA for the indicated time intervals. Radiolabeled lipidic metabolites extracted separately from the cells and cell culture medium were subjected to two-dimensional TLC and visualized by fluorography as described under "Experimental Procedures." Data shown are representative plates from three separate experiments. TG, triglyceride; MAG, monoacylglycerol; SM, sphingomyelin; PS/PI, phosphatidylserine/phosphatidylinositol.

 
Chlorinated Fatty Acids—For fatty acid analyses, cells treated with 2-Cl-[d4]-HDA and [d4]-chlorinated fatty acids were first TLC-purified and subsequently subjected to LC-MS as described above for the synthesis of 2-Cl-16:0 FA. For these analyses, fatty acid molecular species were resolved using a Supelco Discovery® HS C18 5 µ column (15 cm x 2.1 mm) as the stationary phase at a flow rate of 0.2 ml/min. The 2-Cl-16:0 FA was detected by SRM as described above, and [d4]-16:0 FA was detected by SIM m/z 259 in the negative ion mode (electrospray needle voltage = 3.5 kV, capillary T = 320 °C).

Chlorinated Fatty Acid Residues in Phosphatidylcholine—TLC-purified phosphatidylcholine (PC), from lipid extracts of cells treated with either 2-Cl-[3H]-HDA or 2-Cl-[d4]-HDA, was incubated with lipase from Rhizopus arrhizus (Sigma) for 1 h at 37°C in a bilayer consisting of 0.1 M borate buffer (pH 6.5) and ethyl ether. Lipase reaction products were then extracted by the Bligh-Dyer method and were purified using silica gel 60 TLC plates with a mobile phase composed of petroleum ether/ethyl ether/acetic acid (70/30/1, v/v/v). TLC-purified reaction products were first visualized by fluorography. Silica associated with regions of the TLC plate containing radioactivity (visualized by fluorography) were subsequently scraped and quantified by scintillation spectroscopy. For experiments employing stable isotope labeling, bands corresponding to authentic FA and {alpha}-ClFA were extracted and analyzed by LC-MS for [d4]-16:0 FA and [d4]2-Cl-16:0 FA content. Additionally, radiolabeled TLC-purified PC was subjected to either base or acid methanolysis. The resultant 2-ClFAME and FAME was analyzed by TLC and fluorography.


Figure 3
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  		FIGURE 3.
Temporal course of 2-ClHDA incorporation into HCAEC lipids. HCAEC were incubated with 1 µM 2-Cl-[3H]-HDA, and the cellular lipids were extracted at the indicated time points and subsequently subjected to one-dimensional TLC and analyzed as described under "Experimental Procedures." A and B are the neutral and polar lipids, respectively, which were separated in two different solvent systems as described under "Experimental Procedures." Values represent the mean ± S.D. for three independent experiments. Under some conditions, the S.D. was within the size of the symbol for the mean. MAG, monoacylglycerol; CE, cholesterol ester; SM, sphingomyelin; PS/PI, phosphatidylserine/phosphatidylinositol.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Synthetic 2-Cl-[3H]-HDA—2-Cl-[3H]-HDA was synthesized, purified, and used to elucidate {alpha}-chloro fatty aldehyde metabolism in both HCAEC and neutrophils. Fig. 1A demonstrates that synthetic 2-Cl-[3H]-HDA migrates to the same region as that of unlabeled authentic 2-ClHDA. Furthermore, conversion of the synthetic 2-Cl-[3H]-HDA as well as authentic 2-ClHDA to their dimethylacetal derivatives or their pentafluorobenzyl oxime derivatives further demonstrated the purity of the radiolabel. Additionally, Fig. 1B is an overdevelopment of a fluorograph of the synthetic 2-Cl-[3H]-HDA, which demonstrates the purity of this radiolabeled aldehyde.

Metabolism of 2-Cl-[3H]-HDA in HCAEC—HCAEC were incubated with cell culture medium containing 1 µM 2-Cl-[3H]-HDA for either 1 min, 3 or 24 h, and the incorporation of the radiolabel into the lipid metabolites was analyzed by two-dimensional TLC (Fig. 2). These experiments demonstrated that 2-ClHDA was taken up rapidly by HCAEC, and radiolabel derived from 2-ClHDA was eventually incorporated into complex glycerolipids in the HCAEC, including phosphatidylcholine and phosphatidylethanolamine. Radiolabel in these complex glycerolipids remained in the HCAEC and was not released. Additionally, radiolabel derived from 2-ClHDA was found in the {alpha}-chloro fatty alcohol ({alpha}-ClFOH) and {alpha}-chloro fatty acid ({alpha}-ClFA) pools of the cells, as well as these same lipid pools in the cell culture medium (Fig. 2). 2-Cl-[3H]-HDA was not metabolized by cell culture medium without cells present (data not shown). Thus, these results revealed that radiolabeled 2-ClHDA is readily oxidized and reduced to fatty acids and fatty alcohols, respectively, in the cells and that these metabolites are released from the cells. It is likely that the radiolabeled fatty acid is incorporated into the complex glycerolipids. The temporal course of the incorporation of radiolabel from 2-Cl-[3H]-HDA into cellular lipid pools is shown in greater detail in Fig. 3. For these experiments, lipid classes were separated by one-dimensional TLC using mobile phases that separate either neutral or polar lipid classes. After 2-Cl-[3H]-HDA enters the HCAEC, it is rapidly reduced to {alpha}-ClFOH within the first hour of labeling (Fig. 3, inset). Radiolabel also is found in the {alpha}-ClFA pool within the first hour. Fig. 3B shows the incorporation of the radioactivity derived from 2-Cl-[3H]-HDA into the polar lipid pools. The majority of the radioactivity in the polar lipid pools is associated with phosphatidylcholine, and this appears after the first hour of radiolabeling, which suggests that 2-Cl-[3H]-HDA is first oxidized to fatty acids prior to its incorporation into phosphatidylcholine. It should be noted that the migrations of {alpha}-ClFA and {alpha}-ClFOH using TLC are considerably different from non-chlorinated FA and FOH. The assignment of radiolabel to {alpha}-ClFA and {alpha}-ClFOH is based on the co-migration of radiolabel during TLC with regions of authentic {alpha}-ClFA and {alpha}-ClFOH. Using a mobile phase of petroleum ether/ethyl ether/acetic acid (70/30/1) and a silica gel 60 solid phase, the RF for FA and {alpha}-ClFA are 0.34 and 0.24, respectively. The attenuation of {alpha}-ClFA migration on TLC compared with FA is likely because of the electron-withdrawing properties of the {alpha}-chlorine on the carbanion of the carboxylic group, thus reducing its pKa.

Additional studies were performed to determine the metabolism of 2-ClHDA within the range of concentrations that have been observed in neutrophils (24). Fig. 4 shows that the temporal course of 0.1, 1, and 10 µM 2-ClHDA metabolism in HCAEC is very similar. At each concentration of 2-ClHDA, radiolabel was incorporated into both the cellular {alpha}-ClFOH and {alpha}-ClFA pools followed by the incorporation of radiolabel into complex glycerolipids. At 10 µM 2-ClHDA compared with 0.1 and 1 µM 2-ClHDA, there was increased radiolabel in the cellular 2-ClHDA pool over the first hour of labeling, and more label was incorporated into sphingomyelin.

Pulse-chase experiments demonstrated that radiolabel derived from 2-Cl-[3H]-HDA is found predominantly in the cellular {alpha}-ClFOH pool throughout the 1-day chase interval (following a 30-min pulse labeling interval) (Fig. 5A). {alpha}-ClFA is also found in the cell during the chase interval but at lower levels compared with {alpha}-ClFOH (Fig. 5A). The predominant polar lipids that contain radiolabel during the chase interval are the choline glycerophospholipids and the serine (or inositol) glycerophospholipids (Fig. 5B). The predominant radiolabel released into the cell culture medium during the chase interval is {alpha}-ClFOH (Fig. 5C).


Figure 4
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  		FIGURE 4.
2-ClHDA metabolism at selected concentrations of substrate. HCAEC were incubated with either 0.1, 1, or 10 µM 2-Cl-[3H]-HDA for selected intervals as described under "Experimental Procedures." Neutral and polar lipids were analyzed as described under "Experimental Procedures." Values represent the mean ± S.D. for three independent experiments. Under some conditions, the standard deviation was within the size of the symbol for the mean. MAG, monoacylglycerol; CE, cholesterol ester; SM, sphingomyelin; PS/PI, phosphatidylserine/phosphatidylinositol.

 
Metabolism of 2-Cl-[3H]-HDA in Human Neutrophils—Because neutrophils produce 2-ClHDA when activated, 2-Cl-[3H]-HDA metabolism was assessed in stimulated and unstimulated neutrophils. Similar to HCAEC, radiolabel derived from 2-Cl-[3H]-HDA is incorporated into the {alpha}-ClFOH and {alpha}-ClFA pools of the neutrophils soon after uptake into the neutrophils (Fig. 6). Within 1 h, the radiolabel appears in the phosphatidylcholine pool. The predominant difference between 2-ClHDA metabolism in unstimulated and stimulated neutrophils was observed following 15 min of radiolabeling with 2-ClHDA. Less radioactivity was found in the {alpha}-ClFA and {alpha}-ClFOH pools in the stimulated neutrophils as compared with the unstimulated neutrophils (Fig. 6A). Also, following 15 min of concomitant labeling and phorbol 12-myristate 13-acetate stimulation, there is more radiolabeled 2-ClHDA in the medium in comparison to the unstimulated cells (Fig. 6B). It is likely that these differences between the stimulated and unstimulated cells is due to the production of endogenous 2-ClHDA in the stimulated neutrophils that dilutes the radiospecific activity of the endogenously added 2-ClHDA. This suggests that exogenously added 2-Cl-[3H]-HDA has access to compartments shared with endogenously produced 2-ClHDA and that the metabolism of the radiolabel is likely similar to that of endogenously produced 2-ClHDA during neutrophil activation.

Metabolite Identification and Confirmation—To further confirm that {alpha}-ClFOH and {alpha}-ClFA are metabolites of {alpha}-ClFALD, additional studies were performed employing stable isotope labeling of HCAEC and mass spectrometric techniques. Fig. 7B shows the mass spectrum of the PFB ester of the {alpha}-ClFOH, 2-chlorohexadecanol. The parent ion (m/z 470) is accompanied by an ion (m/z 472) that is approximately one-third its intensity, demonstrating that this is a monochlorinated molecule. The GC-MS chromatogram of this derivative is shown in Fig. 7A ({alpha}-ClFOH-standard) using SIM for m/z 470. The PFB ester of 2-chlorohexadecanol is resolved from its non-halogenated analog (FOH-standard, SIM m/z 436). HCAEC that are not treated with 2-ClHDA produce hexadecanol (e.g. m/z 436) but do not produce 2-chlorohexadecanol (e.g. m/z 470) (Fig. 7). In contrast, HCAEC that are treated with 2-ClHDA produce 2-chlorohexadecanol as well as hexadecanol (cells + 2-ClHDA, Fig. 7). As predicted from radiolabeling experiments, 2-chlorohexadecanol is also released into the cell culture medium in cells treated with 2-ClHDA (medium + 2-ClHDA, Fig. 7). Only 2-Cl-[d4]-hexadecanol (e.g. m/z 474), but not 2-chlorohexadecanol (e.g. m/z 470) or [d4]-hexadecanol (e.g. m/z 440), were produced in HCAEC treated with 2-Cl-[d4]-HDA (cells + 2-Cl-[d4]-HDA, Fig. 7). 2-Cl-[d4]-hexadecanol was also released into the cell culture medium from these cells. Additionally, 2-Cl-[d4]-HDA-treated HCAEC produced stable isotope-labeled {alpha}-ClFA. Fig. 8 shows results from LC-MS of stable isotope-labeled FA and {alpha}-ClFA. [d4]-Hexadecanoic acid was monitored by SIM of m/z 259 and [d4]2-chlorohexadecanoic acid was monitored by the SRM transition from m/z 293 to 257 (loss of HCl) (e.g. Fig. 8B). Only cells treated with 2-Cl-[d4]-HDA produced [d4]2-chlorohexadecanoic acid, which was cell-associated as well as released into the medium (Fig. 8).


Figure 5
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  		FIGURE 5.
Pulse-chase 2-Cl-[3H]-HDA radiolabeling of HCAEC. HCAEC were subjected to pulse-chase radiolabeling conditions as described under "Experimental Procedures." Radiolabel associated with each lipid pool was quantified by liquid scintillation spectrometry following one-dimensional TLC as described under "Experimental Procedures." Values shown are from indicated chase interval times in the neutral lipid pool from cells (A), polar lipids from cells (B), and neutral lipids from the cell culture medium (C). Radiolabel was not detected in the polar lipid pool of cell culture medium under these conditions. Values represent the mean ± S.D. for three independent experiments. Under some conditions the standard deviation was within the size of the symbol for the mean. MAG, monoacylglycerol; SM, sphingomyelin; PS/PI, phosphatidylserine/phosphatidylinositol.

 
Because radiolabeling experiments with 2-Cl-[3H]-HDA resulted in the incorporation of radiolabel into PC, further experiments were performed to characterize the radiolabeled moiety that was incorporated into PC. Lipase treatment of TLC-purified radiolabeled PC from HCAEC incubated with 2-Cl-[3H]-HDA resulted in the release of both radiolabeled fatty acid and radiolabeled {alpha}-ClFA (Fig. 9A). Similarly, derivatization of the radiolabeled PC pool by acid methanolysis resulted in the production of the FAME of both FA and 2-Cl-FA (Fig. 9B). Base methanolysis resulted only in the production of radiolabeled FAME (non-chlorinated). Subsequent analysis of authentic {alpha}-ClFA revealed that chlorine is lost from the FAME during base methanolysis but not during acid methanolysis. Parallel experiments were performed with HCAEC treated with 2-Cl-[d4]-HDA. Lipase-treated PC from HCAEC incubated with 2-Cl-[d4]-HDA was subjected to TLC to isolate released fatty acids and {alpha}-ClFA from the PC. Fig. 9, C and D, shows that HCAEC treated with 2-Cl-[d4]-HDA contained PC molecular species containing {alpha}-ClFA as well as non-chlorinated fatty acid derived from the 2-Cl-[d4]-HDA.


Figure 6
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  		FIGURE 6.
Temporal course of 2-ClHDA incorporation into neutrophil lipids. Human neutrophils (5 x 106) were incubated with 1 µM 2-Cl-[3H]-HDA in the presence or absence of 200 nM phorbol 12-myristate 13-acetate (PMA) as described under "Experimental Procedures." Cellular and cell culture medium lipids were extracted at the indicated times and subsequently subjected to one-dimensional TLC and analyzed as described under "Experimental Procedures." A shows the radioactivity incorporated into each lipid class by the neutrophil was quantified for three independent experiments from three different donors. B shows summarized data for radioactivity associated with neutral lipids that are released from neutrophils. Values represent the mean ± S.D. for three independent experiments. MAG, monoacylglycerol; TG, triglyceride, CE, cholesterol ester.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
{alpha}-Chloro fatty aldehydes accumulate in both infarcted myocardium and atherosclerotic lesions (17, 18). Understanding the catabolism of {alpha}-ClFALD is critical in determining the role of this aldehyde in cardiovascular disease. The present studies have demonstrated that 2-ClHDA is readily oxidized and reduced to {alpha}-ClFA and {alpha}-ClFOH, respectively, by HCAEC. In fact, within 30 min of exposure of HCAEC to 2-ClHDA, the intracellular content of 2-ClFOH is greater than that of its precursor, 2-ClHDA. It should also be appreciated that {alpha}-ClFOH and {alpha}-ClFA are produced within the cell but are exported out of the cells. These results are in agreement with previous analyses of the metabolism of non-halogenated fatty acids and fatty alcohols, which are metabolites of fatty acid-fatty alcohol cycling pathways with fatty aldehyde as an intermediate (35). Furthermore, the export of palmitoyl alcohol and palmitic acid out of cells has also been demonstrated in palmitic acid-fed cells (35, 46). Due to the toxicity of fatty aldehydes to cells, it is likely that this pathway functions to protect the cells. It should be noted that the physiological concentrations of 2-ClHDA used in these studies were below cytotoxic concentrations (≥50 µM, data not shown). The demonstration that {alpha}-ClFALD is converted to {alpha}-ClFA and {alpha}-ClFOH suggests that these metabolites may have important roles in mediating the pathophysiological sequelae of myocardial infarction and atherosclerosis. Alternatively, reduction or oxidation of {alpha}-ClFALD may represent important mechanisms that protect tissues from further damage by the aldehyde.

The results herein are the first to demonstrate the presence of {alpha}-ClFA and {alpha}-ClFOH as biological metabolites of {alpha}-ClFALD. These metabolites were identified by multiple independent analyses. Both {alpha}-ClFA and {alpha}-ClFOH were first identified by the co-migration on TLC of radiolabel from HCAEC labeled with 2-Cl-[3H]-HDA with authentic standards. It was important to compare the migration of these metabolites with authentic chlorinated fatty acid and fatty alcohol, because chlorination of these neutral lipid classes alters their migration on TLC compared with non-chlorinated fatty acid and fatty alcohol. Additionally, {alpha}-ClFOH was identified in HCAEC that were incubated either with 2-ClHDA (not labeled) or with 2-Cl-[d4]-HDA. For these experiments, {alpha}-ClFOH was identified by GC-MS analyses of their PFB esters. {alpha}-ClFA was also identified in HCAEC that were incubated with 2-Cl-[d4]-HDA using LC and electrospray ionization-mass spectrometry detection. Taken together, these studies used a combination of radiolabeling techniques and stable isotope labeling coupled with fluorography of TLC plates and mass spectrometric techniques to demonstrate the presence of {alpha}-ClFA and {alpha}-ClFOH metabolites of {alpha}-ClFALD.


Figure 7
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  		FIGURE 7.
GC-MS analyses of fatty alcohol metabolites of 2-ClHDA in HCAEC. HCAEC were treated with either 2-ClHDA, 2-Cl-[d4]-HDA or no additions for 24 h as indicated. PFB esters of TLC-purified fatty alcohol and {alpha}-ClFOH were prepared from cell and cell culture medium lipid extracts as described under "Experimental Procedures." PFB esters of the fatty alcohols were detected by negative ion chemical ionization using GC-MS and compared with authentic PFB ester standards of 2-chloro-hexadecanol (2-Cl-16:0-OH) and hexadecanol (16:0-OH) analyzed separately as indicated. Chromatograms are shown for each indicated condition in A with detection by SIM for 16:0-OH (m/z 436), [d4]-16:0-OH (m/z 440) 2-Cl-16:0-OH (m/z 470), and [d4]2-Cl-16:0-FOH (m/z 474). The mass spectrum for the PFB ester of 2-Cl-16:0-OH is shown in B. Std, standard.

 


Figure 8
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  		FIGURE 8.
Liquid chromatography-mass spectrometry analyses of fatty acid metabolites of 2-ClHDA in HCAEC. HCAEC were treated with either 2-Cl-[d4]-HDA or no additions for 24 h as indicated. Cellular and cell culture medium lipid extracts (as indicated) were subjected to reversedphase LC, and fatty acids were detected by electrospray ionization-mass spectrometry in the negative ion mode as described under "Experimental Procedures." Chromatograms are shown for each indicated condition in A with detection by either SIM for [d4]-16:0 FA (m/z 259) (tr = 13.13 min) or SRM m/z 293 -> 257 for [d4]2-Cl-16:0 FA (tr = 13.78 min). The CID mass spectrum of [d4]2-Cl-16:0 FA standard (negative ion mode) is shown in B. std, standard.

 


Figure 9
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  		FIGURE 9.
2-ClHDA-derived chlorinated fatty acids are incorporated into phosphatidylcholine (PC). TLC-purified PC from HCAEC incubated with 1 µM 2-Cl-[3H]-HDA for 24 h was either treated with lipase (A) or subjected to methanolysis (base or acid methanolysis, as indicated, in B) with reaction products purified by TLC (petroleum ether/ethyl ether/acetic acid (70/30/1 v/v/v)). C and D show LC-MS analysis of lipase-treated PC from HCAEC incubated with 2-Cl-[d4]-HDA for 24 h. SIM m/z 259 ([d4]-16:0 FA, tr = 13.13 min) and SRM 293 -> 257 (loss of HCl from [M-H]– of [d4]2-Cl-16:0 FA, tr = 13.78 min). std, standard.

 
LC-MS analysis of lipase-treated PC extracted from cells treated with 2-Cl-[d4]-HDA revealed that chlorinated fatty acids derived from 2-ClHDA are incorporated into polar lipids such as PC. The incorporation of {alpha}-ClFA into PC indicates that {alpha}-ClFA is converted to its acyl-CoA counterpart. The presence of {alpha}-Cl-fatty acyl-CoA in cells suggests that chlorinated fatty acids are incorporated into other lipids. It is speculated that the substitution of a chlorine atom for a hydrogen atom at the {alpha} carbon may perturb normal membrane dynamics.

2-ClHDA elicits ventricular dysfunction and cardiac injury and is a neutrophil chemoattractant (18, 24). Additionally, physiological levels of 2-ClHDA inhibit eNOS protein expression in human umbilical venous endothelial cells (47). Mechanisms that have been proposed for the loss of eNOS protein, in response to 2-ClHDA, are through mRNA destabilization as well as changes in protein trafficking. Previous studies have shown that 2-ClHDA forms Schiff base adducts with free amines (45). Those studies suggest that one mechanism for alterations in eNOS trafficking might be due to 2-ClHDA associated with the protein. Alternatively, the present data suggest that 2-ClHDA effects on eNOS expression could also be mediated by the {alpha}-ClFA or {alpha}-ClFOH metabolites of {alpha}-ClFALD (2-ClHDA). Importantly, direct effects of 2-ClHDA on eNOS or indirect effects by {alpha}-ClFA or {alpha}-ClFOH metabolites could contribute to pathophysiological changes that may lead to endothelial dysfunction and tissue injury seen in the early phases of atherosclerosis.

Plasmalogens are the precursors of {alpha}-ClFALD that are released by RCS targeting the vinyl ether-linked aliphatic group attached to the sn-1 carbon of the glycerol backbone. In normal cells that are not subject to RCS attack, the metabolic turnover of the vinyl ether-linked aliphatic group of plasmalogens is ~300 times slower than that of the sn-2-esterified fatty acid and the sn-3 polar head group of plasmalogens (42). The biosynthesis of the vinyl ether bond of plasmalogens is preceded by the biosynthesis of an alkyl ether bond linking the sn-1 aliphatic group to the glycerol backbone. The initial step in the biosynthesis of the alkyl ether bond is through an exchange reaction of fatty alcohol with fatty acid esterified to dihydroxyacetone phosphate (48). The biosynthesis of cellular fatty alcohol is through the fatty acid-fatty alcohol cycle (35). It is fascinating that this RCS mechanism that has been shown to chemically target the vinyl ether bond is accompanied by a catabolic mechanism that removes the {alpha}-ClFALD and produces a {alpha}-ClFOH that potentially could have an impact on ether-linked lipid biosynthesis. Under the conditions employed in the present study, radiolabel from 2-Cl-[3H]-HDA was only minimally detected in the sn-1 chain of ether-linked lipids, suggesting that the halogenated alcohol is not incorporated into ether-linked lipids. Furthermore, it is possible that {alpha}-ClFOH inhibits ether-linked lipid biosynthesis. If {alpha}-ClFOH inhibits ether-linked lipid biosynthesis, then the attack of plasmalogens by RCS could have a dual effect in decreasing the content of this critical membrane lipid through the targeting of the vinyl ether and long term effects through the production of the inhibitor {alpha}-ClFOH of plasmalogen biosynthesis. The effects of {alpha}-ClFOH on plasmalogen metabolism remain to be elucidated.

The recent discovery that the plasmalogen vinyl ether bond is targeted by RCS resulting in the production of {alpha}-ClFALD has led to the hypothesis that this mechanism may have a role in ischemia-reperfusion injury and atherosclerosis. It now seems likely that the targeting of plasmalogens by RCS could have multiple effects on host tissues. One effect would be on the membrane dynamics of the membrane that loses plasmalogens by RCS targeting. Another effect would be from Schiff base adduct formation with {alpha}-ClFALD and primary amines in the membrane domain from which the {alpha}-ClFALD originates. Additionally, previous studies have shown {alpha}-ClFALD and lysophosphatidylcholine may have roles in the chemoattraction and tethering of phagocytes to regions of inflammation (17, 24). The present study now suggests there is a family of chlorinated lipidic metabolites produced from {alpha}-ClFALD, and the elucidation of their biological effects may provide important insights into the critical role of plasmalogen oxidation by RCS in MPO-mediated cardiovascular disease.


   FOOTNOTES
 
* This research was supported by National Institutes of Health Grants HL74214 and RR19232 (to D. A. F.) and a predoctoral fellowship (to K. R. W.) and Grant-in-aid 0650044Z (to D. A. F.) from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104. Tel.: 314-977-9264; Fax: 314-977-9205; E-mail: fordda{at}slu.edu.

2 The abbreviations used are: MPO, myeloperoxidase; RCS, reactive chlorinating species; HOCl, hypochlorous acid; FA, fatty acid; {alpha}-ClFA, {alpha}-chloro fatty acid; {alpha}-Cl-FALD, {alpha}-chloro fatty aldehyde; {alpha}-ClFOH, {alpha}-chloro fatty alcohol; 2-ClHDA, 2-chlorohexadecanal; HCAEC, human coronary artery endothelial cells; GC-MS, gas chromatographymass spectrometry; LC-MS, liquid chromatography-mass spectrometry; SIM, selected ion monitoring; SRM, selected reaction monitoring; PC, phosphatidylcholine; PFB, pentafluorobenzoyl; FAME, fatty acid methyl ester; eNOS, endothelial nitric-oxide synthase; TLC, thin layer chromatography. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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J. Lessig, J. Schiller, J. Arnhold, and B. Fuchs
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