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Originally published In Press as doi:10.1194/jlr.D500006-JLR200 on August 16, 2005

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Journal of Lipid Research, Vol. 46, 2506-2513, November 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition

Ichiro Koshiishi1,*, Kazunori Tsuchida*, Tokuko Takajo* and Makiko Komatsu{dagger}

* Nihon Pharmaceutical University, Ina-machi, Kita-Adachi-gun, Saitama 362-0806, Japan
{dagger} Applied Biosystems Japan, Ltd., Chuo-ku, Tokyo 104-0032, Japan

Published, JLR Papers in Press, August 16, 2005. DOI 10.1194/jlr.D500006-JLR200

1 To whom correspondence should be addressed. e-mail: ikoshi{at}nichiyaku.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Lipid alkyl radicals generated from polyunsaturated fatty acids via chemical or enzymatic H-abstraction have been a pathologically important target to quantify. In the present study, we established a novel method for the quantification of lipid alkyl radicals via nitroxyl radical spin-trapping. These labile lipid alkyl radicals were converted into nitroxyl radical-lipid alkyl radical adducts using 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (Cm{Delta}P) (a partition coefficient between octanol and water is approximately 3) as a spin-trapping agent. The resulting Cm{Delta}P-lipid alkyl radical adducts were determined by HPLC with postcolumn online thermal decomposition, in which the adducts were degraded into nitroxyl radicals by heating at 100°C for 2 min. The resulting nitroxyl radicals were selectively and sensitively detected by electrochemical detection.

With the present method, we, for the first time, determined the lipid alkyl radicals generated from linoleic acid, linolenic acid, and arachidonic acid via soybean lipoxygenase-1 or the radical initiator 2,2'-azobis(2,4-dimethyl-valeronitrile).

Abbreviations: AMVN, 2,2'-azobis(2,4-dimethyl-valeronitrile); Cm{Delta}P, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl; ECD, electrochemical detection; epoxy-C18:2, linoleate epoxyallyl radical; LC/MS/MS, liquid chromatography-tandem mass spectrometry

Supplementary key words spin-trapping • high-performance liquid chromatography • lipoxygenases


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Peroxidation of polyunsaturated fatty acids, which are components of cellular membrane and lipoproteins, leads to vital damage to several types of cells. Reactive oxygen/nitrogen species (1), such as hydroxyl radical, hypochlorous acid (2–4), peroxynitrite (5, 6), and singlet oxygen molecule (7–9) are involved in the peroxidation of polyunsaturated fatty acids in the inflammatory lesions. On the other hand, lipoxygenases are speculated to be involved in the peroxidation of polyunsaturated fatty acids in the atheromatous plaque (10). Commonly, lipid alkyl radicals seem to be necessarily produced as an intermediate in these reactions.

To detect labile free radicals via ESR, spin-trapping techniques have been established, in which unstable radicals react with spin-trapping agents to form relatively stable radical adducts (11). To date, nitrone spin-trapping agents, including {alpha}-[4-pyridyl-1-oxide]N-tert-butyl nitrone and 5,5'-dimethyl-1-pyroline-N-oxide, have been used to detect these lipid-derived radicals (12–17). Furthermore, Mason and coworkers (13–15) established an HPLC/ESR system to identify the resulting radical adducts. However, the trapping efficiency of these nitrone compounds toward lipid alkyl radicals is comparatively low. Consequently, these spin-trapping agents are not applicable to the quantification of lipid alkyl radicals.

Five- or six-membered cyclic nitroxyl radicals are relatively stable. Nitroxyl radicals appeared to possess an ability to scavenge carbon-centered radicals (18). This special character of nitroxyl radicals makes it possible to quantitatively trap carbon-centered radicals. Johnson, Caron, and Blough (19) used hydrophilic 3-aminomethyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl to evaluate carbon-centered radical generation: in this method, the adducts were fluorometrically detected after derivatization of amino groups by fluorescamine.

The nitroxyl radical-carbon-centered radical adducts are generally labile at higher temperature, yielding corresponding nitroxyl radicals (20). Therefore, the resulting nitroxyl radicals should become a reporter substance. In the present study, we established a method for the determination of nitroxyl radical-lipid-derived carbon-centered radical adducts by the combination of HPLC and thermal decomposition.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Materials
Soybean lipoxygenase-1 (type I-b), linoleic acid, linolenic acid, arachidonic acid, and linolelaidic acid were purchased from Sigma. 2,2'-Azobis(2,4-dimethyl-valeronitrile) (AMVN) was obtained from Wako Pure Chemicals (Osaka, Japan). 3-Carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (Cm{Delta}P) was purchased from Aldrich Chemical Co., Inc. The nitroxyl radicals were recrystallized in ethanol before use. TSKgel ODS-80Ts QA and TSKguardgel ODS-80Ts were purchased from Tosoh Co. (Tokyo, Japan). Chelex® 100 Resin (100–200 mesh) was purchased from Bio-Rad Laboratories. All other chemicals were of reagent grade.

Lipid-derived radical trapping by nitroxyl radicals
Lipid-derived radicals generated in the polyunsaturated fatty acid/lipoxygenase system were trapped with nitroxyl radicals as follows. Twenty microliters of 2 mM polyunsaturated fatty acid emulsion in 0.1 M phosphate buffer (pH 7.4; treated with Chelex 100) containing 2% ethanol was mixed with 10 µl of 4 mM nitroxyl radical in 0.1 M phosphate buffer (pH 7.4; treated with Chelex 100) and 10 µl of 4 µM soybean lipoxygenase-1 in 0.1 M phosphate buffer (pH 7.4; treated with Chelex 100) in a glass vial tube with screw cap (inner volume, 0.5 ml), and the solution was left to stand at room temperature (25–28°C) for 10 min. The reaction solution was mixed with 160 µl of cold acetonitrile, and then the solution was centrifuged at 10,000 g for 5 min. The supernatant was subjected to HPLC.

Lipid-derived radicals generated chemically from linoleic acid and linolelaidic acid were trapped with Cm{Delta}P as follows. One milliliter of acetonitrile containing 10 mM linoleic acid (or linolelaidic acid), 10 mM Cm{Delta}P, and 10 mM AMVN was transferred into a brown vial tube (inner volume, 2.0 ml) with screw cap, and the vial was left to stand at 37°C for 48 h. The reaction solution was subjected to HPLC.

HPLC analyses of lipid-derived radical-nitroxyl radical adducts
The HPLC assembly consisted of a HPLC pump (PU-2080; Jasco), a sample injector (7725; Rheodyne), a ultraviolet/visible spectrophotometer (L-2420; Hitachi), and a chromatointegrator (Chromatocorder 21; SIC). The chromatographic conditions for the quantification of lipid-derived radical-nitroxyl radical adduct were as follows: TSKgel ODS-80Ts QA column (4.6 mm inner diameter x 150 mm) with TSKguardgel ODS-80Ts guard column (3.2 mm inner diameter x 15 mm); eluent, 75% acetonitrile containing 40 mM acetate buffer (pH 3.0); flow rate, 1.0 ml/min; column temperature, 25–28°C; detection, at 210 nm.

The online thermal decomposition system consisted of a reaction coil (Teflon tube; 0.5 mm inner diameter x 10 m), a dry reaction bath (RO-2061; Jasco), and a back pressure coil (Teflon tube; 0.25 mm inner diameter x 5 m). The system was followed by electrochemical detection (ECD) (ECD-300; Eicom).

The online liquid chromatography-tandem mass spectrometry (LC/MS/MS) system consisted of the Agilent 1100 HPLC system and Q TRAP® LC/MS/MS system (Applied Biosystems/MDS Sciex, Concord, Ontario, Canada) equipped with an electrospray ion source. The liquid chromatograph flow rate (1.0 ml/min) into the mass spectrometer inlet was adjusted to 200 µl/min with a splitter.

Quantification of lipid-derived radical-nitroxyl radical adducts by thermal decomposition
Reaction solution containing carbon-centered alkyl radical-Cm{Delta}P adducts was diluted twice with acetonitrile. The solution was transferred into a glass vial with screw cap and heated at 100°C. The solutions were subjected to ESR spectrometry. The conditions for the ESR measurements were as follows: microwave power, 10 mW; frequency, 9.430 GHz; magnetic field, 339.5 ± 7.5 mT; modulation, 100 kHz, 0.2 mT; time constant, 0.3 s.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Thermal decomposition of nitroxyl radical-lipid alkyl radical adducts
The active ferric form of lipoxygenase can catalyze the abstraction of hydrogen from the bis-allylic carbon atom of the polyunsaturated fatty acid in a stereospecific manner, yielding a pentadienyl radical complexed with the ferrous enzyme (21). At lower oxygen concentrations, nitroxyl radical traps the radical intermediate on the enzyme, yielding the ferrous form of lipoxygenase. If hydroperoxy fatty acid coexists in this reaction system, the ferrous form of lipoxygenase is converted into the active ferric form, generating alkoxyl radical (22–27). This oxygen-centered alkoxyl radical is immediately converted into carbon-centered epoxyallyl radical in aqueous solution through intramolecular rearrangement (28, 29). Nitroxyl radical can trap these carbon-centered radicals, generating equimolar amounts of the nitroxyl radical-lipid alkyl radical adducts and the nitroxyl radical-lipid epoxyallyl radical adducts. The overall paths are shown in Scheme 1
Scheme 1. Possible reaction paths in the linoleic acid/lipoxygenase/3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (Cm{Delta}P) system. An excess amount of linoleic acid over oxygen content is present in this system.

.

When 0.1 M phosphate buffer (pH 7.4; treated with Chelex 100) containing 1 mM linoleic acid, 1 µM soybean lipoxygenase-1, and 200 µM Cm{Delta}P was left to stand in a glass vial tube with screw cap at room temperature for 10 min, the residual Cm{Delta}P in the reaction system was <2 µM. The reaction solution was diluted five times with acetonitrile, and then the solution was centrifuged at 10,000 g for 10 min. The generation of nitroxyl radical-lipid alkyl radical (m/z 463) and nitroxyl radical-lipid epoxyallyl radical (m/z 479) in the reaction system was confirmed by LC/MS/MS analysis, as shown in Fig. 1.



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  		Fig. 1. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) analyses of nitroxyl radical-linoleate carbon-centered radical adducts. Forty microliters of 0.1 M phosphate buffer (pH 7.4) containing 1.0 mM linoleic acid, 1.0 mM Cm{Delta}P, and 1.0 µM soybean lipoxygenase-1 was left to stand in a glass vial tube with screw cap (inner volume, 0.5 ml) at room temperature for 10 min. The reaction solution was mixed with 160 µl of acetonitrile, and then the solution was centrifuged at 10,000 g for 5 min. The supernatant was subjected to LC/MS/MS. A: Extracted Ion Chromatogram (XIC) of m/z 479 ([M+H]+) from LC/MS data of Cm{Delta}P-linoleate carbon-centered radical adducts. B: XIC of m/z 463 from LC/MS data of Cm{Delta}P-linoleate carbon-centered radical adducts. C: MS/MS of protonated molecule of m/z 479 in MS of adduct 1 (6.38 min). D: MS/MS of protonated molecule of m/z 479 in MS of adduct 2 (7.01 min). E: MS/MS of protonated molecule of m/z 463 in MS of adduct 3 (13.02 min). F: MS/MS of protonated molecule of m/z 463 in MS of adduct 5 (17.58 min). It should be noted that the nitroxyl radical-linoleate alkyl radical adducts degrade into hydroxylamine (m/z 185 for Cm{Delta}P) in the collision cell of the mass spectrometer.

 
The supernatant of the reaction solution was further diluted twice with water. A portion of the solution (0.2 ml) was transferred into a glass vial with screw cap, and the vial was heated at 100°C. After a certain time interval, ESR spectra of regenerated Cm{Delta}P in the solution were measured. The time course of the relative recovery of Cm{Delta}P is shown in Fig. 2. It is remarkable that the thermal decomposition of Cm{Delta}P-lipid-derived radical adducts proceeded in two phases: early and late decomposition. As shown in Fig. 2 (inset), curve-fitting evaluation indicates that ~50% of Cm{Delta}P-lipid-derived radical adducts degraded rapidly, and the other 50% degraded gradually. The labile adduct was estimated to degrade by >80% within 2 min at 100°C.



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  		Fig. 2. Thermal decomposition of Cm{Delta}P-linoleate carbon-centered radical adducts into the corresponding nitroxyl radical. Phosphate buffer (0.1 M; pH 7.4) containing 1.0 mM linoleic acid, 200 µM Cm{Delta}P, and 1.0 µM soybean lipoxygenase-1 was left to stand in a glass vial tube with screw cap (inner volume, 0.5 ml) at room temperature for 10 min. The residual Cm{Delta}P was <1% of initial Cm{Delta}P content. The solution was diluted twice with acetonitrile, and then the solution was heated at 100°C. The solution was subjected to X-band ESR. The ESR conditions are described in Experimental Procedures. Error bars represent standard deviation.

 
As shown in Fig. 2, Cm{Delta}P-lipid-derived radical adducts were completely degraded into Cm{Delta}P by heating at 100°C for 2 h. Four Cm{Delta}P-lipid-derived radical adducts were fractionated by HPLC, and then each adduct was standardized via the thermal decomposition method using Cm{Delta}P as a standard substance (Fig. 3). Approximately 96% of adducts were recovered through the fractionation. The resulting standard solutions of Cm{Delta}P-lipid-derived radical adducts should be left to stand at –20°C.



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  		Fig. 3. Preparation and standardization of Cm{Delta}P-linoleate carbon-centered radical adducts produced in the linoleic acid/lipoxygenase/Cm{Delta}P system. Forty microliters of 0.1 M phosphate buffer (pH 7.4) containing 1.0 mM linoleic acid, 0.2 mM Cm{Delta}P, and 1.0 µM soybean lipoxygenase-1 was left to stand in a glass vial tube with screw cap (inner volume, 0.5 ml) at room temperature for 10 min. The reaction solution was mixed with 160 µl of acetonitrile, and then the solution was centrifuged at 10,000 g for 5 min. The supernatant (100 µl; a 20 µl aliquot was injected in each fractionation) was subjected to HPLC with ultraviolet detection. Each adduct was fractionated, and then the fractions were dried under argon gas. The residues were suspended in 50% acetonitrile. A part of the suspension was heated at 100°C for 2 h, and then the solutions were subjected to ESR spectrometry. AU, absorbance units; LA-OOH, hydroperoxy linoleic acid; LA, linoleic acid.

 
Postcolumn thermal decomposition HPLC with ECD capacity for the detection of nitroxyl radical-lipid alkyl radical adducts
Based on this finding, we established HPLC with thermal decomposition, in which the regenerated nitroxyl radical was selectively and sensitively detected by ECD. A diagram of the HPLC apparatus is shown in Scheme 2
Scheme 2. Diagram of the HPLC/electrochemical detection (ECD) system with online postcolumn thermal decomposition. i.d., inner diameter.

.

For the quantitative spin-trapping, 0.1 M phosphate buffer (pH 7.4) containing 1.0 mM linoleic acid, 1 µM soybean lipoxygenase-1, and 1.0 mM Cm{Delta}P was left to stand at room temperature for 10 min, and then the solution was diluted five times with cold acetonitrile. After centrifugation at 10,000 g for 5 min, the supernatant was subjected to HPLC with ultraviolet detection at 210 nm. The chromatogram is shown in Fig. 4A. Approximately equal amounts of four adducts were detected: 9-Cm{Delta}P-10E,12Z-C18:2, 13-Cm{Delta}P-9Z,11E-C18:2, and two regioisomers of nitroxyl radical-linoleate epoxyallyl radical adducts (Cm{Delta}P-epoxy-C18:2). Similarly, the supernatant was subjected to HPLC-ECD with postcolumn thermal decomposition. The chromatogram is shown in Fig. 4B. Remarkably, the nitroxyl radical-lipid alkyl radical adducts 9-Cm{Delta}P-10E,12Z-C18:2 and 13-Cm{Delta}P-9Z,11E-C18:2 were detected. However, in this system, Cm{Delta}P-epoxy-C18:2 content was extremely low. Through these findings, it appeared that nitroxyl radical-lipid epoxyallyl radical adducts are comparatively resistant to thermal decomposition. Figure 5 shows the correlation between the applied potential and the relative response. To quantitatively trap lipid alkyl radicals, we estimated an optimal nitroxyl radical content. As shown in Fig. 6, Cm{Delta}P content >0.5 mM was required for the quantification of linoleate alkyl radicals generated in the linoleic acid/lipoxygenase/Cm{Delta}P system.



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  		Fig. 4. Chromatographic analyses of Cm{Delta}P-linoleate carbon-centered radical adducts. A: HPLC with ultraviolet detection at 210 nm. B: HPLC/ECD with online postcolumn thermal decomposition. The HPLC conditions are described in Experimental Procedures. AU, absorbance units.

 


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  		Fig. 5. Effect of applied potential on the electrochemical detection of the Cm{Delta}P-linoleate alkyl radical adducts. The reaction solution of the linoleic acid/lipoxygenase/Cm{Delta}P system was subjected to HPLC/ECD with online postcolumn thermal decomposition. The relative response (%) represents the relative peak area against that detected at 950 mV versus Ag/AgCl.

 


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  		Fig. 6. Effect of Cm{Delta}P content on the spin-trapping of linoleate alkyl radical. Forty microliters of 0.1 M phosphate buffer (pH 7.4) containing 1.0 mM linoleic acid, Cm{Delta}P, and 1.0 µM soybean lipoxygenase-1 was left to stand in a glass vial tube with screw cap (inner volume, 0.5 ml) at room temperature for 10 min. The reaction solution was mixed with 160 µl of acetonitrile, and then the solution was centrifuged at 10,000 g for 5 min. The supernatant was subjected to HPLC/ECD with online postcolumn thermal decomposition (800 mV vs. Ag/AgCl).

 
Quantitative evaluation of chemically and enzymatically generated lipid alkyl radicals from polyunsaturated fatty acid
Linoleic acid (9Z,12Z-C18:2), linolenic acid (9Z,12Z,15Z-C18:3), and arachidonic acid (5Z,8Z,11Z,14Z-C20:4) are the principal polyunsaturated fatty acids found in organisms. Lipoxygenases are believed to convert fatty acids into specific oxidation products. In the present study, we evaluated the lipid alkyl radical generation from these individual polyunsaturated fatty acids and their mixture via soybean lipoxygenase-1. The chromatograms are shown in Fig. 7. In common, the generation of two regioisomers of Cm{Delta}P-lipid alkyl radical adducts from each individual fatty acid was confirmed. Nitroxyl radical competes with the oxygen molecule for lipid alkyl radical on soybean lipoxygenase-1. Consequently, Cm{Delta}P-lipid alkyl radical adducts should resemble hydroperoxy fatty acids generated at lower oxygen content in regiospecificity and stereospecificity. Regarding the regiospecificity of hydroperoxy fatty acids generated in polyunsaturated fatty acid/soybean lipoxygenase-1 systems (30–32), the regiospecificity and stereospecificity of Cm{Delta}P-lipid alkyl radical adducts were identified as follows: 13-Cm{Delta}P-9Z,11E-C18:2 (major) and 9-Cm{Delta}P-10E,12Z-C18:2 (minor) from linoleic acid; 13-Cm{Delta}P-9Z,11E,15Z-C18:3 (major) and 9-Cm{Delta}P-10E,12Z,15Z-C18:3 (minor) from linolenic acid; 15-Cm{Delta}P-5Z,8Z,11Z,13E-C20:4 (major) and 12-Cm{Delta}P-5Z,8Z,10E,14Z-C20:4 (minor) from arachidonic acid. The satisfactory separation of these adducts was achieved by reversed-phase HPLC, as shown in Fig. 7D.



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  		Fig. 7. Chromatographic evaluation of lipid alkyl radical generation from linoleic acid (9Z,12Z-C18:2), linolenic acid (9Z,12Z,15Z-C18:3), arachidonic acid (5Z,8Z,11Z,14Z-C20:4), or a mixed sample via soybean lipoxygenase-1. One millimolar linoleic acid (A), 1.0 mM linolenic acid (B), 1.0 mM arachidonic acid (C), and a mixed sample (0.33 mM each; D) were treated with 1.0 µM soybean lipoxygenase-1 in the presence of 1.0 mM Cm{Delta}P. The HPLC conditions are described in Experimental Procedures. Detection was at 800 mV versus Ag/AgCl. Sample volume was 10 µl (A–C) and 15 µl (D).

 
In a similar manner, we tried to trap lipid alkyl radicals, which were chemically generated from linoleic acid or linolelaidic acid via the lipophilic radical initiator AMVN. One milliliter of acetonitrile containing 10 mM linoleic acid or linolelaidic acid, 10 mM Cm{Delta}P, and 10 mM AMVN [half-life at 37°C, 90 h (33)] was left to stand in a glass vial with screw cap (inner volume, 2 ml) at 37°C for 48 h. The reaction solutions were diluted 20 times with acetonitrile, and the solutions were subjected to HPLC. The chromatograms are shown in Fig. 8. Two main adducts (m/z 463) were detected in both systems. The intermediate carbon-centered E/Z-pentadiene radical undergoes resonance stabilization into two positionally isomeric pentadiene radicals, C-9 and C-13 positions. In a nonenzymatic reaction, the cis double bond of the radicals can spontaneously isomerize to a thermodynamically favored trans configuration (34). Consequently, despite the difference in the stereospecificity of precursor fatty acids, the stereospecificities of the resulting Cm{Delta}P-lipid alkyl radical adducts were identical.



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  		Fig. 8. Chromatographic analyses of Cm{Delta}P-lipid alkyl radical adducts generated from linoleic acid (9Z,12Z-C18:2) and linolelaidic acid (9E,12E-C18:2) via the radical initiator 2,2'-azobis(2,4-dimethyl-valeronitrile) (AMVN). Acetonitrile solution containing 10 mM Cm{Delta}P, 10 mM AMVN, and 10 mM linoleic acid (A) or linolelaidic acid (B) was left to stand at 37°C for 48 h. One volume of the reaction solution was mixed with 19 volumes of acetonitrile, and a 10 µl portion of the solution was subjected to HPLC/ECD with online postcolumn thermal decomposition (800 mV vs. Ag/AgCl).

 
We succeeded, for the first time, in establishing an HPLC system for the sensitive and selective quantification of lipid alkyl radicals generated through chemical and enzymatic hydrogen abstraction. The HPLC system can be easily assembled from commercially available equipment in scientific laboratories. We expect that this method will be applied to the assessment of lipid alkyl radical generation in biological systems.

Manuscript received February 18, 2005 and in revised form July 1, 2005 and in re-revised form July 28, 2005.


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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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