Advertisement
J. Lipid Res.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1194/jlr.M600327-JLR200 on August 30, 2006

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M600327-JLR200v1
47/11/2515    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lawson, J. A.
Right arrow Articles by Rokach, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lawson, J. A.
Right arrow Articles by Rokach, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Journal of Lipid Research, Vol. 47, 2515-2524, November 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Oxidized derivatives of {omega}-3 fatty acids: identification of IPF3{alpha}-VI in human urine1

John A. Lawson*, Seongjin Kim{dagger}, William S. Powell§, Garret A. FitzGerald* and Joshua Rokach2,{dagger}

* Institute for Translational Medicine and Therapeutics and Department of Pharmacology, The University of Pennsylvania, Philadelphia, PA 19104
{dagger} Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology, Melbourne, FL 32901
§ Meakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec, Canada H2X 2P2

1 The iP nomenclature used throughout this manuscript was previously reported (Rokach, J., S. P. Khanapure, S. W. Hwang, M. Adiyaman, J. A. Lawson, and G. A. FitzGerald. 1997. Prostaglandins. 54: 853–873). Another nomenclature is also in use (Taber, D. F., J. D. Morrow, and L. J. Roberts II. 1997. Prostaglandins. 53: 63–67). Back

Published, JLR Papers in Press, August 30, 2006.

2 To whom correspondence should be addressed. e-mail: jrokach{at}fit.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Isoprostanes (iPs) are prostaglandin-like molecules derived from autoxidation of polyunsaturated fatty acids (PUFAs). Urinary iP levels have been used as indices of in vivo lipid peroxidation. Thus far, it has only been possible to measure iPs derived from arachidonic acid in urine, because levels of iPs/neuroprostanes (nPs) derived from {omega}3-PUFAs have been found to be below detection limits of available assays. Because of the interest in {omega}3-PUFA dietary supplementation, we developed specific methods to measure nPF4{alpha}-VI and iPF3{alpha}-VI [derived from 4,7,10,13,16,19-docosahexaenoic acid (DHA) and 5,8,11,14,17-eicosapentaenoic acid (EPA)] using a combination of chemical synthesis, gas chromatography/mass spectrometry (GC/MS), and liquid chromatography tandem mass spectrometry (LC/MS/MS). Although nPF4{alpha}-VI was below the detection limit of the assay, we conclusively identified iPF3{alpha}-VI in human urine by GC/MS and LC/MS/MS. The mean levels in 26 subjects were ~300 pg/mg creatinine. Our failure to detect nPF4{alpha}-VI may have been due to its rapid metabolism by ß-oxidation to iPF3{alpha}-VI, which we showed to occur in rat liver homogenates. In contrast, iPF3{alpha}-VI is highly resistant to ß-oxidation in vitro. Thus iPF3{alpha}-VI can be formed by two mechanisms: i) direct autoxidation of EPA, and ii) ß-oxidation of nPF4{alpha}-VI, formed by autoxidation of DHA. This iP may therefore serve as an excellent marker for the combined in vivo peroxidation of EPA and DHA.

Supplementary key words isoprostanes • arachidonic acid • eicosapentaenoic acid • docosahexaenoic acid • neuroprostane F4{alpha}-VI • mass spectrometry • lipid peroxidation • {omega}3-polyunsaturated fatty acids

Abbreviations: AA, arachidonic acid; AD, Alzheimer's disease; BDS, Base Deactivated Silica; DHA, 4,7,10,13,16,19-docosahexaenoic acid; ECNI, electron capture/negative ion; EPA, 5,8,11,14,17-eicosapentaenoic acid; iP, isoprostane; i.s., internal standard; LC, liquid chromatography; MS/MS, tandem mass spectrometry; nP, neuroprostane; PFB, pentafluorobenzyl; SPE, solid-phase extraction; TMS, trimethylsilyl


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Isoprostanes (iPs) are a class of natural products generated by the action of free radicals on polyunsaturated fatty acids (PUFAs) (1, 2). We have previously reported on the total synthesis of iPs and metabolites derived from free radical peroxidation of arachidonic acid (AA) (3–7). These synthetic markers have been used to discover and characterize four classes of iPs in biological fluids (2, 8, 9). The availability of these standards permitted the development of gas chromatography/mass spectrometry (GC/MS) and liquid chromatography tandem mass spectrometry (LC/MS/MS) methodologies to examine the distribution of iPs within each of these classes (10, 11). These methods have been used to measure iPs, in particular Groups III and VI, in biological fluids, and have revealed a correlation between their levels and the severity of diseases such as atherosclerosis (12) and Alzheimer's disease (AD) (13).

Although iPs can be formed from many PUFAs, research to date has focused almost exclusively on F2-iPs derived from AA. 5,8,11,14,17-Eicosapentaenoic acid (EPA) and 4,7,10,13,16,19-docosahexaenoic acid (DHA) are two other naturally occurring PUFAs that constitute an important and sizeable component of cellular phospholipids, especially in the case of DHA, which is found in high levels in the brain (14). Because of the increased absorption of EPA and DHA by individuals whose diets include substantial amounts of fatty fish, iPs and neuroprostanes (nPs) derived from these {omega}3-PUFAs may be more abundant than those derived from {omega}6-PUFAs in these subjects. Moreover, because of their health benefits, {omega}3-PUFAs are increasingly being used as dietary supplements and have been the subject of various clinical trials (15). For these reasons, it has become very important to be able to assess the extent of peroxidation of these highly unsaturated fatty acids. However, attempts to measure {omega}3-derived iPs/nPs have so far met with only limited success, especially with noninvasively collected samples such as urine. Although F3-iPs are formed following chemical peroxidation of EPA (16), a recently published study failed to identify these substances in tissues from rodents and humans on chow and typical North American diets, respectively (17). In rodents, it was only possible to detect tissue F3-iPs following dietary supplementation with EPA (17). F4-nPs are formed by chemical oxidation of DHA (18, 19) and are found in human and rodent brain tissue (19). However, it has thus far not been possible to detect these substances in urine from humans, including subjects with AD (20).

Further investigation into the biological occurrence of {omega}3-iPs and nPs and the identification of selective markers for these substances is therefore of great importance. We have previously shown that iPF2{alpha}-VI is a more abundant marker of endogenous AA peroxidation than the more commonly measured iPF2{alpha}-III (8-iso-PGF2{alpha}) (Fig. 1 ) (2, 5). The objective of the current study was to determine whether the analogous EPA- and DHA-derived oxidation products, iPF3{alpha}-VI and nPF4{alpha}-VI, could serve as markers for peroxidation of {omega}3-PUFAs. We succeeded in detecting for the first time high levels of iPF3{alpha}-VI in human urine, but were unable to detect nPF4{alpha}-VI. This is probably due to the rapid ß-oxidation of this substance; we found that it is readily converted to iPF3{alpha}-VI by rat liver homogenates. Thus, iPF3{alpha}-VI could serve as an excellent in vivo marker for {omega}3-PUFAs, because it can be formed as a result of peroxidation of both EPA and DHA.


Figure 1
View larger version (4K):
[in this window]
[in a new window]
  		Fig. 1. Structures of PGF2{alpha}, iPF2{alpha}-VI, iPF3{alpha}-VI, and nPF4{alpha}-VI.

 

   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Synthesis of iPF3{alpha}-VI
Details of the total synthesis of iPF3{alpha}-VI 3 and its deuterated analog are shown in Figs. 2 and 3. Synthon 7 was prepared from commercially available methyl-5-chloro-5-oxo-valerate (21). The synthesis of the aldehyde 6 was performed in 10 steps from 5 as described by us previously for the synthesis of nPF4{alpha}-VI (21). The structure of iPF3{alpha}-VI was confirmed by 1H NMR (CDCl3): {delta} 5.67–5.25 (m, 6H), 4.12–3.98 (m, 3H), 2.82 (m, 3H), 2.31 (m, 2H), 2.12–1.90 (m, 6H), 1.54 (m, 3H), 1.23 (m, 2H), 0.89 (t, 3H), and by electron impact MS [methyl ester, trimethylsilyl (TMS) deriv]: m/z 582(M+), 567(M-15), 492(M-90) base peak, 403(M-179), 391(M-191).


Figure 2
View larger version (6K):
[in this window]
[in a new window]
  		Fig. 2. Synthesis of iPF3{alpha}-VI. Reagents and conditions: 7, LiHMDS, THF, 6, –78°C, 3 h, 68% (a); S-BINAL-H, THF, –100°C, 4 h, 94% (b); 6:3:1 = THF:formic acid:H2O, room temperature, 3 h, 74% (c); and 5% KOH, THF, room temperature, 2 h, 97% (d).

 

Figure 3
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 3. Synthesis of [19,19,20,20-2H]iPF3{alpha}-VI. Reagents and conditions: t-BuOK, THF, –20°C, 5 h, 82% (a); periodinane, CH2Cl2, room temperature, 2 h, 97% (b); 7, LiHMDS, THF, 14, –78°C, 14 h, 76% (c); S-BINAL-H, THF, –100°C, 5 h, 97% (d); TBDMSCl, Im, CH2Cl2, room temperature, 8 h, 75% (e); Me2AlCl, CH2Cl2, –20°C to room temperature, 3 h, 72% (f); periodinane, t-BuOH, CH2Cl2, room temperature, 5 h, 95%, (g); 19, LiHMDS, HMPA, THF, 18, –78°C, 3 h, 68% (h); 6:3:1 = THF:formic acid:H2O, room temperature, 3 h, and KOH, THF, room temperature, 1.5 h, 85%, two steps (i).

 
Synthesis of [19,19,20,20-2H]iPF3{alpha}-VI
[19,19,20,20-2H]iPF3{alpha}-VI 21, the tetradeuterated analog of iPF3{alpha}-VI, was prepared as shown in Fig. 3. The deuterated synthon 19, which was used to complete the bottom side chain, was prepared from propargyl alcohol in five steps (21). We have recently reported the transformation of 11 to 14 in connection with the total synthesis of [21,21,22,22-2H]nPF4{alpha}-VI (21). The structure of d4-iPF3{alpha}-VI was confirmed by 1H NMR (CD3OD): {delta} 5.52–5.25 (m, 6H), 3.98 (q, 1H, J = 4.3 and 10.9), 3.92 (q, 1H, J = 7 and 11.8), 3.83 (q, 1H, J = 6.8 and 11.2), 2.74 (m, 2H), 2.63 (m, 2H), 2.43 (m, 1H), 2.25 (m, 2H, J = 6.8), 2.02 (m, 2H), 1.58 (m, 3H), 1.46 (m, 2H), 1.21 (s, 1H).

ß-Oxidation of nPF4{alpha}-VI and iPF3{alpha}-VI by rat liver homogenates
Liver from Brown Norway rats was minced in ice-cold 0.25 M sucrose and then diluted with this medium to give a final ratio of 5 ml/g tissue. The suspension was homogenized by hand using six to eight strokes in a Potter-Elvehjem homogenizer in an ice bath, and the homogenate was centrifuged at 600 g for 10 min. To aliquots (1 ml) of the supernatant fraction were added 1 ml of 100 mM phosphate buffer, pH 7.4, containing 50 mM KCl, 7.2 mM MgCl2, 8 mM ATP, 1 mM CoA, 4 mM L-carnitine, and 4 mM NAD+. Either nPF4{alpha}-VI [final concentration, 20 µM; synthesized as described previously (21)] or iPF3{alpha}-VI (final concentration, 20 µM) was added, and the mixture was incubated for 90 min at 37°C. The incubations were stopped by the addition of 1 vol of MeOH and cooled on ice. Water was then added to give a final concentration of 15% MeOH, and the mixture was centrifuged. The supernatant was subjected to solid-phase extraction (SPE) as described previously (22) using a C18 SepPak cartridge (Waters Associates, Milford, MA) that had been pretreated with MeOH and water. The cartridge was eluted successively with 15% MeOH, water, and petroleum ether prior to elution of eicosanoids with methyl formate. The methyl formate was then removed using a rotary evaporator, and the residue was dissolved in a small volume of ethanol prior to further analysis.

Processing of urine samples for MS analysis
Urine was collected from 20 healthy individuals, evenly divided between males and females. Samples were immediately frozen at –80°C until further processing. After thawing, 10 ng [2H4]nPF4{alpha}-VI [synthesized as described previously (21)] was added to 2 ml aliquots, which were mixed and allowed to equilibrate for 15 min at room temperature. The sample was extracted by SPE on a StrataX cartridge, (Phenomenex, Torrance, CA), which had been conditioned with 1 ml of acetonitrile (MeCN) followed by 1 ml H2O. The sample was then applied to the cartridge, which was washed successively with H2O (1 ml) and 5% MeCN in H2O (1 ml), dried by applying a vacuum for 15 min, and eluted with 1 ml of 5% MeCN in EtOAc. The eluate was dried under a gentle stream of nitrogen, and the sample was stored in 40 µl MeCN at –80°C until analysis, at which time H2O (160 µl) was added and the sample was filtered in a Costar Spin-X HPLC filter (0.2 µm nylon; Corning Inc., Corning, NY).

iPF3{alpha}-VI (Group I)
Urine was obtained from six healthy individuals, three males and three females. Samples were immediately frozen at –80°C until further processing. After thawing, 2 ml aliquots were taken, and to each was added 6.9 ng of [2H4]iPF3{alpha}-VI and 10 ng of the methoxime derivative of 2,3-dinor-6-keto-PGF1{alpha} (d6k-PGF1{alpha}-MO). The samples were mixed, allowed to equilibrate for 15 min at room temperature, and extracted by SPE on StrataX cartridges as described above. The eluate was dried under a gentle stream of nitrogen, dissolved in 1 ml 75% EtOAc in hexane, and applied to a diol SPE cartridge (Bond Elut 2OH; Varian, Palo Alto, CA), which was washed with 1 ml of 75% EtOAc in hexane and eluted with 1 ml of 5% MeCN in EtOAc containing 0.1% acetic acid. Samples were then dried under nitrogen and dissolved in 100 µl of 10% MeCN in water for injection onto a 150 x 2 mm HyperClone 5 µm C18 Base Deactivated Silica (BDS) column (Phenomenex, Torrence, CA) using a linear gradient between 10% and 50% solvent B over 20 min at a flow rate of 200 µl/min. Solvent A was water-acetic acid (100:0.005), adjusted to pH 5.7 with ammonium hydroxide, whereas solvent B was MeCN-MeOH-acetic acid (95:5:0.005). The transition monitored was m/z 370 -> 150 (d6k-PGF1{alpha}-MO). The elution time of d6k-PGF1{alpha}-MO served to predict the elution time of iPF3{alpha}-VI. One minute after the apex of the d6k-PGF1{alpha}-MO peak, the column eluate was diverted from the mass spectrometer and collected for a period of 0.6 min. Samples were split into two equal portions for quantitation of iPF3{alpha}-VI by GC/MS and LC/MS/MS as described below.

iPF3{alpha}-VI (Group II)
Urine from 20 healthy individuals (see above) was thawed, 1 ml aliquots were removed, and to each was added 7.5 ng of [2H4]iPF3{alpha}-VI. iPF3{alpha}-VI was then extracted by SPE as described above for nPF4{alpha}-VI, and the extract was dissolved in MeCN (20 µl) and stored at –80°C until analysis by LC/MS/MS as described below.

The urine samples were obtained with the informed consent of the donors.

GC/MS
GC/MS was done on a Hewlett-Packard 5973 mass spectrometer equipped with a chemical ionization source coupled to an HP 6890 gas chromatograph. The instrument was operated in the electron capture/negative ion (ECNI) mode, using ammonia as the moderating gas. The stationary phase was a column of 60 m x 0.25 mm i.d. DB-5MS (0.25 µm; Agilent, Palo Alto, CA). The split/splitless injector was operated at 260°C and the interface at 280°C. The temperature program consisted of 1 min at 190°C followed by a linear increase to 300°C at a rate of 30°C/min.

LC/MS/MS
All LC/MS/MS was performed using a Thermo-Finnigan TSQ Quantum Ultra AM (Thermo Electron Corp.), except for the Group I study on iPF3{alpha}-VI, which was performed using a Micromass Ultima instrument (Waters). In both cases, the mass spectrometer was interfaced with two Shimadzu LC-10ADvp pumps (Shimadzu, Columbia, MD), a Shimadzu SCL-10Avp system controller, and a CTC Analytics HTC PAL autosampler (LEAP Technologies, Carrboro, NC). All LC/MS/MS analyses were performed in the negative-ion electrospray mode using Argon, 1.5 mTorr, as the collision gas. Quantitation was by peak area ratios. Mobile phases for HPLC were as described above. The flow rate was 0.2 ml/min. All LC conditions and MS/MS tuning parameters were optimized by injection or infusion of authentic synthetic standards.

LC/MS/MS quantitation of nPF4{alpha}-VI from urine Filtered samples (see above) were injected onto a 150 x 2 mm HyperClone 3 µm C18 BDS column (Phenomenex). The mobile phase was a gradient between 20% and 32% B over 20 min. The transitions monitored were m/z 377 -> 113 for endogenous nPF4{alpha}-VI and m/z 381 -> 113 for d4-nPF4{alpha}-VI. Collision energy was 21 V. Source collision-induced dissociation was 10 eV. Capillary temperature was 400°C, and spray voltage was 4 kV.

LC/MS/MS quantitation of iPF3{alpha}-VI from urine (Group I) Filtered urine samples were injected onto a 150 x 2 mm Luna 5 µm C18(2) column (Phenomenex). The mobile phase was 23% B at a flow rate of 0.2 ml/min. The transitions monitored were m/z 351 -> 115 for endogenous iPF3{alpha}-VI and m/z 355 -> 115 for the tetradeuterated analog. Collision energy was 21 V. Cone potential was –81 V, source temperature was 70°C, desolvation temperature was 200°C, and capillary potential was 3 kV (Fig. 7a).


Figure 7
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 7. Identification of iPF3{alpha}-VI in urine by LC/MS/MS and GC/MS. IPs were extracted from a urine sample from a subject in Group I to which d4-iPF3{alpha}-VI had been added as an internal standard (i.s.). iPF3{alpha}-VI was purified by HPLC using d6k-PGF1{alpha}-MO as a marker for its elution position as described in Methods (initial study). A: LC/MS/MS electrospray-selected reaction monitoring analysis of iPF3{alpha}-VI. Transitions characteristic of d4-iPF3{alpha}-VI (m/z 355 -> 115) and iPF3{alpha}-VI (m/z 351 -> 115) are shown in the upper and lower panels, respectively. B: ECNI GC/MS analysis of iPF3{alpha}-VI in the above urinary extract following conversion to its PFB ester, TMS ether derivative. The ions due to loss of PFB are shown in the upper (m/z 571, d4-iPF3{alpha}-VI) and lower (m/z 567, iPF3{alpha}-VI) panels, respectively.

 
LC/MS/MS quantitation of iPF3{alpha}-VI from urine (Group II) Filtered samples were injected onto a 150 x 2 mm HyperClone 3 µm C18 BDS column. The mobile phase was a gradient between 20% and 32% B over 20 min at a flow rate of 0.2 ml/min. The transitions monitored were m/z 351 -> 115 for endogenous iPF3{alpha}-VI and m/z 355 -> 115 for the tetradeuterated analog. MS conditions were as described above for nPF4{alpha}-VI. These conditions were also used for the analysis of ß-oxidation products of nPF4{alpha}-VI.

GC/MS analysis of nPF4{alpha}-VI metabolites Samples were converted to the pentafluorobenzyl (PFB) ester by adding 10 µl N,N-diisopropylethylamine (Sigma Chemical Co., St. Louis, MO) and 20 µl 10% PFB Br (Sigma) in MeCN. After 10 min at room temperature, the solvent was removed under a stream of nitrogen and the TMS ether derivative was formed by treatment with 10 µl pyridine (Sigma) and 10 µl bis(trimethylsilyl)trifluoroacetamide (Supelco, Bellefonte, PA) for 10 min. Samples were then dried under nitrogen and dissolved in dodecane (Sigma) for injection into the GC/MS. Partial mass spectra were obtained by scanning the mass range from m/z 300 to m/z 600 for 0.4 s. Selected ion monitoring (SIM) techniques were also used, focusing on m/z 567 for iPF3{alpha}-VI and m/z 571 for the tetradeuterated analog.

GC/MS quantitation of iPF3{alpha}-VI from urine Samples were dried under nitrogen and the PFB ester, TMS ether derivatives were prepared as described above. SIM was used, monitoring m/z 567 for iPF3{alpha}-VI and m/z 571 for the tetradeuterated analog. Quantitation was by peak area ratios.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Synthesis of iPF3{alpha}-VI and [19,19,20,20-2H]iPF3{alpha}-VI
The first total and stereospecific syntheses of iPF3{alpha}-VI 3 and its deuterated analog 21 were accomplished as shown in Figs. 2 and 3. It is interesting to note that in the synthesis of [19,19,20,20-2H]iPF3{alpha}-VI 21, the completion of the bottom side chain was done differently from that of the nondeuterated analog, as can be seen in Fig. 3 (transformation 18 to 20). To spare the deuterated synthon 19, the introduction of the deuterium piece was effected at the last stage. The structures of both compounds were confirmed by NMR. An analysis of the deuterium content of the deuterated analog expressed as the ratio of 2H0/2H4, determined by GC/MS, was 0.0009.

Measurement of nPF4{alpha}-VI in urine
We attempted to measure the levels of nPF4{alpha}-VI in urine samples from healthy subjects by LC/MS/MS, but were unable to detect this nP in any of the 20 samples tested (Fig. 4A ). In contrast, we were easily able to detect a small amount (100 pg/ml) of exogenous synthetic nPF4{alpha}-VI that had been added to the same sample (Fig. 4B). As expected, the tetradeuterated analog had a retention time slightly shorter than that of the unlabeled compound. The retention times of both the labeled and unlabeled compounds were confirmed by separate analysis of chemically synthesized standards. Note that some of the samples exhibited a small peak near the retention time of endogenous nPF4{alpha}-VI. When two other ion transitions characteristic of the fragmentation of nPF4{alpha}-VI were monitored (m/z 377 -> 199 and m/z 377 -> 359), it was clear that this peak did not originate from authentic nPF4{alpha}-VI.


Figure 4
View larger version (13K):
[in this window]
[in a new window]
  		Fig. 4. iPF4{alpha}-VI is not present in detectable amounts in human urine. Urine from a healthy individual was extracted and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS), monitoring the transitions m/z 377 -> 113 (unlabeled nPF4{alpha}-VI) and 381 -> 113 (d4-nPF4{alpha}-VI). A: Mass chromatogram of an extract from a urine sample to which the internal standard d4-nPF4{alpha}-VI (10 ng) was added. B: Mass chromatogram of an identical sample that also contained chemically synthesized unlabeled nPF4{alpha}-VI (100 pg). As shown by the arrows, nPF4{alpha}-VI can only be detected in the sample containing the exogenous material.

 
Conversion of nPF4{alpha}-VI to iPF3{alpha}-VI by ß-oxidation
In view of the presence of DHA and nPs in the brain, we were surprised to find nPF4{alpha}-VI to be undetectable in urine. We speculated that this could be due to its metabolism by ß-oxidation, and to test this hypothesis, we investigated its metabolism by a rat liver homogenate in the presence of ATP, CoA, L-carnitine, and NAD+. Incubation of nPF4{alpha}-VI (20 µM) with this preparation for 90 min led to the formation of a single metabolite, amounting to 19% of the substrate. Analysis by LC/MS revealed the presence of an intense ion with an m/z of 351 (M-1; data not shown). This indicates a loss of 26 mass units from the substrate nPF4{alpha}-VI, which exhibited an intense ion at m/z 377 (M-1), suggesting that it had been converted by ß-oxidation to iPF3{alpha}-VI. Collision-induced dissociation of the ion at m/z 351 gave rise to a series of daughter ions with m/z values of 297, 289, 271, 253, 245, 217, 191, and 115 (base peak) (Fig. 5A ), which was virtually identical to the mass spectrum obtained from synthetic iPF3{alpha}-VI (Fig. 5B).


Figure 5
View larger version (18K):
[in this window]
[in a new window]
  		Fig. 5. Mass spectrum of the ß-oxidation product of nPF4{alpha}-VI. A: The mass spectrum of the major product formed during incubation of nPF4{alpha}-VI with a rat liver homogenate in the presence of ATP, CoA, L-carnitine, and NAD+, as described in Methods. B: The mass spectrum of chemically synthesized iPF3{alpha}-VI.

 
The chromatographic profile of nPF4{alpha}-VI metabolites with the properties of iPF3{alpha}-VI (i.e., intense ion at m/z 351 giving rise to a daughter ion at m/z 115) is shown in the lower panel of Fig. 6A . The retention time (tR) of the major peak (10.8 min) is nearly identical to that of synthetic d4-iPF3{alpha}-VI (10.6 min), shown for comparison in the top panel of Fig. 6A. As expected, the deuterated analog has a very slightly shorter retention time due to its shorter C-2H bonds. A small peak, presumably an isomer of iPF3{alpha}-VI, was also detected. To provide further confirmation of the identity of the major nPF4{alpha}-VI metabolite, we examined its properties by GC/MS after conversion to its PFB ester, TMS ether derivative. One major metabolite with an intense ion at m/z 567 (M-PFB) and a tR of 17.17 min, only slightly longer than that of authentic d4-iPF3{alpha}-VI (m/z 571; tR, 17.10 min), was observed (Fig. 6B).


Figure 6
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 6. Identification of the ß-oxidation product of nPF4{alpha}-VI as iPF3{alpha}-VI by LC/MS/MS and gas chromatography (GC)/MS. A: LC/MS/MS electrospray-selected reaction monitoring analysis of an extract of a rat liver homogenate incubated with nPF4{alpha}-VI as described in Methods. d4-iPF3{alpha}-VI was added as an internal standard (i.s.). The transition m/z 355 -> 115 (d4-iPF3{alpha}-VI) is shown in the upper panel, whereas the transition m/z 351 -> 115 is shown in the lower panel. B: Electron capture/negative ion (ECNI) GC/MS of the same extract shown in A, after conversion of iPs to their pentafluorobenzyl (PFB) ester, and trimethylsilyl (TMS) ether derivatives. The upper panel shows the mass chromatogram for m/z 571 (d4-iPF3{alpha}-VI), whereas the lower panel shows m/z 567 (iPF3{alpha}-VI).

 
In contrast to nPF4{alpha}-VI, iPF3{alpha}-VI was quite resistant to metabolism by ß-oxidation. Following incubation with a rat liver homogenate under conditions identical to those described above and analysis by LC/MS/MS, the only major component observed was the unmetabolized substrate. A very small amount (~0.06% of the substrate) of a substance with the properties expected for 2,3-dinor-iPF3{alpha}-VI was detected, but the amount was insufficient for positive identification.

Identification of iPF3{alpha}-VI in urine
We conducted an initial study with six subjects (Group I) using a rigorous purification protocol, involving an independent LC purification step prior to mass spectrometric analysis to demonstrate conclusively the presence of iPF3{alpha}-VI in human urine. d6k-PGF1{alpha}-MO was added to the urine along with the internal standard d4-iPF3{alpha}-VI to act as a marker to define the elution position of iPF3{alpha}-VI. Samples were then purified by two steps of SPE using reversed-phase and diol stationary phases, followed by LC using MS/MS to detect the d6k-PGF1{alpha}-MO marker. The material eluting immediately after the elution marker was then analyzed by both LC/MS/MS and GC/MS.

Analysis by LC/MS/MS revealed a constituent with an intense ion at m/z 351 (M-1), which gave rise to a daughter ion at m/z 115 (Fig. 7A , lower panel). We previously found that this daughter ion is characteristic of Group VI iPs and does not occur to an appreciable extent with iPs of other groups (10). A single major peak was observed with a tR of 26.1 min, slightly longer than that of the corresponding ion (355 -> 115) from the tetradeuterated internal standard (tR, 25.7 min; Fig. 7A, upper panel), as expected for iPF3{alpha}-VI. The sample was also analyzed by GC/MS following conversion of iPs to their PFB ester, TMS ether derivatives, which would be expected to give rise to a derivative with a major fragment ion at m/z 567 (M-PFB). When this ion was monitored, a major peak was observed with a tR of 13.5 min, slightly longer than that of the internal standard (tR, 13.45 min; Fig. 7B, upper panel), confirming the presence of iPF3{alpha}-VI.

Urinary levels of iPF3{alpha}-VI in healthy subjects
In an initial study, the urinary levels of iPF3{alpha}-VI were measured by both the GC/MS and LC/MS/MS methods in six healthy individuals (Group I), including three males and three females. The levels of iPF3{alpha}-VI were between approximately 200 and 300 pg/ml creatinine for five out of the six subjects, with excellent agreement between the two methods (Fig. 8 ). However, one subject had substantially higher levels of iPF3{alpha}-VI with both methods, and in this case, the level as determined by LC/MS/MS was nearly twice as high as that found using the GC/MS assay (Fig. 8, inset). This could be due to either overestimation of the internal standard in the GC/MS sample or overestimation of the endogenous signal in the LC/MS/MS sample. Because the internal standard is added at a relatively high level, it is a single, symmetrical peak in all samples, and not likely to be artifactually elevated. Considering the endogenous LC/MS/MS traces (i.e., m/z 351 -> 115), five of the six samples exhibited a single peak with a retention time identical to that of authentic iPF3{alpha}-VI. Only the chromatogram of the outlier sample had an additional peak, raising the possibility that the peak coeluting with the internal standard may be somewhat elevated due to the coelution of an interfering compound that was not removed by the sample prepurification.


Figure 8
View larger version (13K):
[in this window]
[in a new window]
  		Fig. 8. Correlation between LC/MS/MS and GC/MS assays for iPF3{alpha}-VI. Following addition of the internal standards, urine samples from the six subjects in Group I were extracted and iPF3{alpha}-VI was purified by HPLC prior to analysis by mass spectrometry, as described in Methods. Five (open circles) out of six individuals had levels of iPF3{alpha}-VI at or below 325 pg/mg creatinine, whereas the sixth individual had considerably higher levels (closed circle, inset).

 
A second study was performed in which the urinary levels of iPF3{alpha}-VI were measured by LC/MS/MS in an additional 20 subjects, including 10 males and 10 females (Group II). The average age was 34 ± 13.6 years, and the average body mass index (weight/height2) was 22.3 ± 2.6. The mean urinary level of iPF3{alpha}-VI among these subjects was 274 ± 201 pg/mg creatinine (mean ± SD). There were no significant differences between males and females (Fig. 9 ) and no correlation of iPF3{alpha}-VI levels with body mass index (data not shown). There was a tendency for older subjects to have higher levels of iPF3{alpha}-VI, but this was not statistically significant (Fig. 9).


Figure 9
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 9. Relationship between gender, age, and urinary iPF3{alpha}-VI levels in healthy human subjects. iPF3{alpha}-VI was quantitated by LC/MS/MS in urine samples from 20 individuals (Group II) as described in Methods. The group consisted of 10 males (closed circles) and 10 females (open circles).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
EPA and DHA are the main PUFAs in fish and fish oil (23) and are popular food supplements, sold in pharmacies alone or in combination with vitamins and minerals. The reason for the interest in these {omega}3-PUFAs stems from observations that populations that consume large amounts of fatty fish, such as the Inuit (24), have a lower incidence of myocardial infarction than populations that consume a Western diet high in {omega}6-PUFAs. The potentially beneficial effects of EPA and DHA have been supported by a variety of epidemiological and interventional studies of fish consumption (15, 25). However, it remains unclear whether the reported benefit reflects a decrease in saturated fat consumption or a beneficial effect of {omega}-3 fatty acids. A quantitative marker of {omega}-3 fatty acid bioavailability that could be related to clinical outcome would assist in the prospective, randomized, controlled trials necessary to evaluate the clinical efficacy of dietary supplements.

Multiple potentially beneficial effects have been ascribed to {omega}-3 fatty acids, including a reduction in blood pressure (26), modulation of the response to endogenous and exogenous thrombolytic agents (27), antiarrhythmic actions (28), inhibition of platelet activation (29), and modulation of inflammation (30), triglycerides, and VLDL (31). Some of these effects are, at least in part, mediated by alterations in the biosynthesis and effects of eicosanoids (25, 32). {omega}3-PUFAs, in particular DHA, are present in high levels in the brain, and may play a role in cognitive development (33). There is also evidence that there is an inverse relationship between the content of EPA/DHA in the diet and the incidence of depression (25). Like AA, EPA and DHA can be converted into a series of iPs, which, in the case of DHA-derived iPs, have been termed nPs (34). However, prior to the present study, no assays to measure the in vivo production of F3-iPs derived from EPA through the measurement of urinary levels were available. Although nPs can be measured in brain tissue, an attempt to measure F4-nPs by GC/MS in the urine of patients suffering from AD found them to be undetectable (20). We selected Group VI F3-iPs and F4-nPs as our initial targets for detection in biological fluids because we had previously observed that among AA-derived iPs, Group VI iPs such as iPF2{alpha}-VI 2 and 8,12-iso-iPF2{alpha}-VI are the most abundant in human urine (2, 8). Because of the close structural analogy of iPF3{alpha}-VI 3 and nPF4{alpha}-VI to iPF2{alpha}-VI 2 (Fig. 1), it would seem likely that this would also be the case for Group VI F3-iPs and F4-nPs.

Because DHA is the most abundant PUFA in the brain, we initially sought to measure urinary nPF4{alpha}-VI levels as an in vivo indicator of its peroxidation. Such biomarkers might be used to titrate the response to {omega}-3s in interventional studies to assess their value in the assessment of cognitive function. For this reason, we recently developed a method for the total synthesis of nPF4{alpha}-VI and its tetradeutero analog (21) to permit its quantitation in biological fluids. However, in the present study, we were unable to detect nPF4{alpha}-VI in urine by LC/MS/MS. One potential reason for this could be that it was rapidly metabolized in vivo. Comparison of the structures of iPF2{alpha}-VI and iPF3{alpha}-VI (Fig. 1) reveals that the carboxyl side chains of these two molecules are identical, whereas the carboxyl side chain of nPF4{alpha}-VI is elongated and contains an addition double bond at C4. Others as well as ourselves have reported that the presence of a hydroxyl group in the 5-position of the carboxyl side chain of leukotrienes and iPs can have a marked effect on their metabolic fates (9, 35, 36). 5-Hydroxyeicosanoids are resistant to ß-oxidation, probably because the 5-hydroxyl group interacts with the carboxyl group, thus limiting access of the enzymes involved in this process. It was this assumption that 5-hydroxy-iPs would be resistant to ß-oxidation that led us to focus originally on AA-derived Group VI iPs, because we suspected that they would be more abundant in urine than the more commonly measured Group III iPs and hence easier to discover and measure. We therefore reasoned that the greater distance between this hydroxyl group and the C1 carboxylic acid group in nPF4{alpha}-VI could result in less protection against ß-oxidation compared with iPF2{alpha}-VI and iPF3{alpha}-VI. We compared the extent of ß-oxidation of iPF3{alpha}-VI and nPF4{alpha}-VI in vitro by rat liver homogenates to test this hypothesis. In contrast to iPF3{alpha}-VI, which was highly resistant to ß-oxidation, nPF4{alpha}-VI was a good substrate for this process, being converted to iPF3{alpha}-VI (i.e., 2,3-dinor-4,5-dihydro-nPF4{alpha}-VI) to the extent of 19%. The identity of this metabolite was established by GC/MS and LC/MS/MS comparison with chemically synthesized standards.

The extent of ß-oxidation of nPF4{alpha}-VI was over three times greater than that of [17,18,19,20-2H4]iPF2{alpha}-III, which has a 7 carbon carboxylic acid side chain without a hydroxyl group. Under identical reaction conditions, only 3.6% of iPF2{alpha}-III was converted to the dinor metabolite, along with an additional 2.2% to the dihydro-dinor compound (data not shown). This suggests that nPF4{alpha}-VI is particularly prone to ß-oxidation at C1, providing a possible explanation for our inability to identify this substance in human urine. Others, too, have been unable to measure nP formation in the urine of AD patients (20). The metabolism argument presented here is strengthened by the finding that iPF2{alpha}-III is metabolized in vivo, with a major (29%) urinary metabolite being the dinor dihydro derivative (37). We and others have also studied this metabolic step (38–40). Because nPF4{alpha}-VI is a much better substrate than iPF2{alpha}-III for ß-oxidation, we would expect its rate of in vivo metabolism to be several-fold higher, hence explaining the lack of nPF4{alpha}-VI in urine. We were unable to identify any dinor-nPF4{alpha}-VI in incubations with rat liver homogenates, indicating that the 4,5-double bond of nPF4{alpha}-VI was completely reduced. This is presumably due to the formation of the {Delta}2,4-conjugated diene 22, followed by reduction to the {Delta}3 metabolite 23 and ß-oxidation (Fig. 10 ) (41). A similar situation has previously been encountered in the metabolism of LTE4 (34, 35, 42), which is metabolized by {omega}-oxidation, followed by two cycles of ß-oxidation coupled with reduction of the 14,15-double bond.


Figure 10
View larger version (6K):
[in this window]
[in a new window]
  		Fig. 10. Proposed metabolism pathway (ß-oxidation) of nPF4{alpha}-VI.

 
The facile metabolism of nPF4{alpha}-VI to iPF3{alpha}-VI suggests that the formation of iPF3{alpha}-VI could not only be an indicator of the production of EPA-derived iPs, but also, to a considerable extent, could reflect the endogenous formation of nPF4{alpha}-VI. For this reason, we prepared iPF3{alpha}-VI and [19,19,20,20-2H]iPF3{alpha}-VI by total synthesis to allow the measurement of the former compound in urine by MS. We used two independent assays based on GC/ECNI-MS and LC/MS/MS to conclusively identify iPF3{alpha}-VI in urine. The requirement for conversion of iPF3{alpha}-VI to a volatile derivative for GC/ECNI-MS analysis adds an additional level of complexity to this method. Thus, with GC/ECNI-MS of the PFB ester, TMS ether derivatives are separated in the gas phase and ionized by the gentle capture of thermal electrons by the electrophilic PFB group, leading to cleavage of the bond between the PFB and carboxyl group, leaving the intact quasi-molecular anion at m/z 567 for MS analysis. The underivatized compounds are chromatographed with LC/MS/MS on a reversed-phase column at a pH that ensures their anionic form. The highly selective nature of the selected reaction monitoring technique screens out all ions other than those possessing m/z ratios equivalent to that of the analyte (m/z 351 for iPF3{alpha}-VI), fragments them by collision with argon, and finally, filters a preselected product ion (m/z 115 for iPF3{alpha}-VI). Despite the differing principles of separation and detection between the GC and LC methods described above, there was very good agreement in the urinary levels of iPF3{alpha}-VI between the two methods, providing conclusive evidence for the identification of this iP.

In contrast to the extensive amount of information available on the biological activities of the enzymatically derived prostaglandins and leukotrienes, relatively little is known about the bioactivities of iPs. Although a limited number of studies have been performed on certain iPs derived from AA, it is not yet known whether iPs derived from EPA and DHA have biological activities. We have shown that exogenous iPF2{alpha}-III can activate the thromboxane receptor in platelets and the vasculature in vivo (43) and that 8,12-iso-iPF2{alpha}-III can activate the receptor for PGF2{alpha} in vitro (44, 45). However, it is unknown whether these iPs function as endogenous receptor ligands at the concentrations formed in vivo. It would be of interest to determine whether iPs derived from EPA and DHA are also biologically active and to compare their effects with those of AA-derived iPs.

The present data raise several important questions. Does the measurement of iPF3{alpha}-VI in urine of normal volunteers reflect two sources of iPs, namely, unmetabolized iPF3{alpha}-VI derived from EPA and the dinor dihydro metabolite of nPF4{alpha}-VI? If this is true, measurement of iPF3{alpha}-VI could be an excellent index of the combined endogenous autooxidation of EPA and DHA, and might reflect a process of relevance to disease progression in syndromes of neurodegeneration, such as AD. Recently, F4-nPs have been detected in brain tissue (19, 46). However, the measurement of changes in nP levels in urine as an index of AD has not been successful (20). On the basis of the present results, it would seem likely that this could be explained by the ß-oxidation of F4-nPs to F3-iPs. A number of studies have demonstrated elevated levels of urinary F2-iPs in AD (47), but because of the high levels of DHA in brain lipids (14), it may be more relevant to measure iPs derived from this PUFA, particularly because they could be relatively selective markers of lipid peroxidation in the brain. Because of the rapid ß-oxidation of nPF4{alpha}-VI to iPF3{alpha}-VI, measurement of the latter substance in urine may provide important mechanistic clues and could be of diagnostic value in the investigation of neurodegenerative diseases such as AD.


   ACKNOWLEDGMENTS
 
The authors are grateful to Sylvie Gravel for assistance with the ß-oxidation experiments. This work was supported by National Institutes of Health Grants HL-81873 (J.R.), HL-69835 (J.R.), and HL-70128 (G.A.F.), Canadian Institutes of Health Research Grant MOP-6254 (W.S.P.), the Heart and Stroke Foundation of Quebec, and the J.T. Costello Memorial Research Fund. J.R. also wishes to acknowledge the National Science Foundation for a Bruker 400 MHz NMR instrument (Grant CHE-03 42251).

Manuscript received July 21, 2006 and in revised form August 25, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
  1. Morrow, J. D., K. E. Hill, R. F. Burk, T. M. Nammour, K. F. Badr, and L. J. Roberts II. 1990. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc. Natl. Acad. Sci. USA. 87: 9383–9387.[Abstract/Free Full Text]

  2. Praticò, D., O. P. Barry, J. A. Lawson, M. Adiyaman, S. W. Hwang, S. P. Khanapure, L. Iuliano, J. Rokach, and G. A. FitzGerald. 1998. IPF2{alpha}-I: An index of lipid peroxidation in humans. Proc. Natl. Acad. Sci. USA. 95: 3449–3454.[Abstract/Free Full Text]

  3. Hwang, S. W., M. Adiyaman, S. Khanapure, L. Schio, and J. Rokach. 1994. Total synthesis of 8-epi-PGF2{alpha}. A novel strategy for the synthesis of isoprostanes. J. Am. Chem. Soc. 116: 10829–10830.[CrossRef]

  4. Adiyaman, M., J. A. Lawson, S. W. Hwang, S. P. Khanapure, G. A. FitzGerald, and J. Rokach. 1996. Total synthesis of a novel isoprostane iPF2{alpha}-I and its identification in biological fluids. Tetrahedron Lett. 37: 4849–4852.[CrossRef]

  5. Adiyaman, M., H. Li, J. A. Lawson, S. W. Hwang, S. P. Khanapure, G. A. FitzGerald, and J. Rokach. 1997. First total synthesis of isoprostane iPF2{alpha}-III. Tetrahedron Lett. 38: 3339–3342.[CrossRef]

  6. Pudukulathan, Z., S. Manna, S. W. Hwang, S. P. Khanapure, J. A. Lawson, G. A. FitzGerald, and J. Rokach. 1998. Diels-Alder approach to isoprostanes. Total synthesis of iPF2{alpha}-V. J. Am. Chem. Soc. 120: 11953–11961.[CrossRef]

  7. Kim, S., Y. Jung, J. A. Lawson, G. A. FitzGerald, and J. Rokach. 2001. iPF2{alpha}-III metabolism. First total synthesis of 2,3-dinor iPF2{alpha}-III, a primary ß-oxidation metabolite. Tetrahedron Lett. 42: 8277–8280.[CrossRef]

  8. Lawson, J. A., H. Li, J. Rokach, M. Adiyaman, S. W. Hwang, S. P. Khanapure, and G. A. FitzGerald. 1998. Identification of two major F2 isoprostanes, 8,12-iso- and 5-epi-8,12-iso-isoprostane F2{alpha}-VI, in human urine. J. Biol. Chem. 273: 29295–29301.[Abstract/Free Full Text]

  9. Adiyaman, M., J. A. Lawson, S. P. Khanapure, G. A. FitzGerald, and J. Rokach. 1998. Total synthesis of 17,17,18,18-d4-iPF2{alpha}-VI and quantification of iPF2{alpha}-VI in human urine by gas chromatography/mass spectrometry. Anal. Biochem. 262: 45–56.[CrossRef][Medline]

  10. Li, H., J. A. Lawson, M. Reilly, M. Adiyaman, S. W. Hwang, J. Rokach, and G. A. FitzGerald. 1999. Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F2-isoprostanes in human urine. Proc. Natl. Acad. Sci. USA. 96: 13381–13386.[Abstract/Free Full Text]

  11. Lawson, J. A., J. Rokach, and G. A. FitzGerald. 1999. Isoprostanes: formation, analysis and use as indices of lipid peroxidation in vivo. J. Biol. Chem. 274: 24441–24444.[Free Full Text]

  12. Praticò, D., R. K. Tangirala, D. J. Rader, J. Rokach, and G. A. FitzGerald. 1998. Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in ApoE-deficient mice. Nat. Med. 4: 1189–1192.[CrossRef][Medline]

  13. Praticò, D., J. Rokach, J. Lawson, and G. A. FitzGerald. 2004. F2-isoprostanes as indices of lipid peroxidation in inflammatory diseases. Chem. Phys. Lipids. 128: 165–171.[CrossRef][Medline]

  14. Sastry, P. S. 1985. Lipids of nervous tissue: composition and metabolism. Prog. Lipid Res. 24: 69–176.[CrossRef][Medline]

  15. Kris-Etherton, P. M., W. S. Harris, L. J. Appel, and for the AHA Nutrition Committee. 2003. Omega-3 fatty acids and cardiovascular disease: new recommendations from the American Heart Association. Arterioscler. Thromb. Vasc. Biol. 23: 151–152.[Free Full Text]

  16. Nourooz-Zadeh, J., B. Halliwell, and E. E. Anggard. 1997. Evidence for the formation of F3-isoprostanes during peroxidation of eicosapentaenoic acid. Biochem. Biophys. Res. Commun. 236: 467–472.[CrossRef][Medline]

  17. Gao, L., H. Yin, G. L. Milne, N. A. Porter, and J. D. Morrow. 2006. Formation of F-ring isoprostane-like compounds (F3-isoprostanes) in vivo from eicosapentaenoic acid. J. Biol. Chem. 281: 14092–14099.[Abstract/Free Full Text]

  18. Fam, S. S., L. J. Murphey, E. S. Terry, W. E. Zackert, Y. Chen, L. Gao, S. Pandalai, G. L. Milne, L. J. Roberts II, N. A. Porter, et al. 2002. Formation of highly reactive A-ring and J-ring isoprostane-like compounds (A4/J4-neuroprostanes) in vivo from docosahexaenoic acid. J. Biol. Chem. 277: 36076–36084.[Abstract/Free Full Text]

  19. Musiek, E. S., J. K. Cha, H. Yin, W. E. Zackert, E. S. Terry, N. A. Porter, T. J. Montine, and J. D. Morrow. 2004. Quantification of F-ring isoprostane-like compounds (F4-neuroprostanes) derived from docosahexaenoic acid in vivo in humans by a stable isotope dilution mass spectrometric assay. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 799: 95–102.[Medline]

  20. Montine, T. J., J. F. Quinn, D. Milatovic, L. C. Silbert, T. Dang, S. Sanchez, E. Terry, L. J. Roberts II, J. A. Kaye, and J. D. Morrow. 2002. Peripheral F2-isoprostanes and F4-neuroprostanes are not increased in Alzheimer's Disease. Ann. Neurol. 52: 175–179.[CrossRef][Medline]

  21. Kim, S., J. A. Lawson, D. Praticò, G. A. FitzGerald, and J. Rokach. 2002. The first total synthesis of iPF4{alpha}-VI and its deuterated analog. Tetrahedron Lett. 43: 2801–2805.[CrossRef]

  22. Powell, W. S. 1980. Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl silica. Prostaglandins. 20: 947–957.[CrossRef][Medline]

  23. Goodnight, S. H., Jr., W. S. Harris, W. E. Connor, and D. R. Illingworth. 1982. Polyunsaturated fatty acids, hyperlipidemia, and thrombosis. Arteriosclerosis. 2: 87–113.[Free Full Text]

  24. Dyerberg, J., and H. O. Bang. 1979. Hemostatic function and platelet unsaturated fatty acids in Eskimos. Lancet. 2: 433–435.[CrossRef][Medline]

  25. Smith, W. L. 2005. Cyclooxygenases, peroxide tone and the allure of fish oil. Curr. Opin. Cell Biol. 17: 174–182.[CrossRef][Medline]

  26. Knapp, H. R., and G. A. FitzGerald. 1989. The antihypertensive effects of fish oil. A controlled study of polyunsaturated fatty acid supplements in essential hypertension. N. Engl. J. Med. 320: 1037–1043.[Abstract]

  27. Braden, G. A., H. R. Knapp, D. J. Fitzgerald, and G. A. FitzGerald. 1990. Dietary fish oil accelerates the response to coronary thrombolysis with tissue-type plasminogen activator. Circulation. 82: 178–187.[Abstract/Free Full Text]

  28. Brouwer, I. A., A. Geelen, and M. B. Katan. 2006. n-3 Fatty acids, cardiac arrhythmia and fatal coronary heart disease. Prog. Lipid Res. 45: 357–367.[CrossRef][Medline]

  29. Knapp, H. R., I. A. Reilly, P. Alessandrini, and G. A. FitzGerald. 1986. In vivo indexes of platelet and vascular function during fish-oil administration in patients with atherosclerosis. N. Engl. J. Med. 314: 937–942.[Abstract]

  30. Basu, A., S. Devaraj, and I. Jialal. 2006. Dietary factors that promote or retard inflammation. Arterioscler. Thromb. Vasc. Biol. 26: 995–1001.[Abstract/Free Full Text]

  31. Connor, W. E. 1988. Effects of omega-3 fatty acids in hypertriglyceridemic states. Thromb. Haemost. 14: 271–284.

  32. Braden, G. A., H. R. Knapp, and G. A. FitzGerald. 1991. Suppression of eicosanoid biosynthesis during coronary angioplasty by fish oil and aspirin. Circulation. 84: 679–685.[Abstract/Free Full Text]

  33. Wainwright, P. E. 2002. Dietary essential fatty acids and brain function: a developmental perspective on mechanisms. Proc. Nutr. Soc. 61: 61–69.[Medline]

  34. Roberts II, L. J., T. J. Montine, W. R. Markesbery, A. R. Tapper, P. Hardy, S. Chemtob, W. D. Dettbarn, and J. D. Morrow. 1998. Formation of isoprostane-like compounds (neuroprostanes) in vivo from docosahexaenoic acid. J. Biol. Chem. 273: 13605–13612.[Abstract/Free Full Text]

  35. Murphy, R. C., and P. Wheelan. 1997. Metabolism of leukotrienes in vitro and in vivo. In SRS-A to Leukotrienes: The Dawning of a New Treatment. S. Holgate and S. E. Dahlen, editors. Blackwell Science Ltd., London. 101–120.

  36. Keppler, D., M. Huber, W. Hagmann, H. A. Ball, A. Guhlmann, and S. Kastner. 1988. Metabolism and analysis of endogenous cysteinyl leukotrienes. Ann. N. Y. Acad. Sci. 524: 68–74.[Medline]

  37. Roberts II, L. J., K. P. Moore, W. E. Zackert, J. A. Oates, and J. D. Morrow. 1996. Identification of the major urinary metabolite of the F2-isoprostane 8-iso-prostaglandin F2{alpha} in humans. J. Biol. Chem. 271: 20617–20620.[Abstract/Free Full Text]

  38. Burke, A., J. A. Lawson, E. A. Meagher, J. Rokach, and G. A. FitzGerald. 2000. Specific analysis in plasma and urine of 2,3-dinor-5,6-dihydro-isoprostane F2{alpha}-III, a metabolite of isoprostane F2{alpha}-III and an oxidation product of {gamma}-linolenic acid. J. Biol. Chem. 275: 2499–2504.[Abstract/Free Full Text]

  39. Kim, S., W. S. Powell, J. A. Lawson, S. H. Jacobo, D. Praticò, G. A. FitzGerald, K. Maxey, and J. Rokach. 2005. iPF2{alpha}-III-17,18,19,20-d4: total synthesis and metabolism. Bioorg. Med. Chem. Lett. 15: 1613–1617.[CrossRef][Medline]

  40. Chiabrando, C., A. Valagussa, C. Rivalta, T. Durand, A. Guy, E. Zuccato, P. Villa, J-C. Rossi, and R. Fanelli. 1999. Identification and measurement of endogenous ß-oxidation metabolites of 8-epi-prostaglandin F2{alpha}. J. Biol. Chem. 274: 1313–1319.[Abstract/Free Full Text]

  41. Schulz, H., and W. H. Kunau. 1987. Beta-oxidation of unsaturated fatty acids: a revised pathway. Trends Biochem. Sci. 12: 403–406.[CrossRef]

  42. Delorme, D., A. Foster, Y. Girard, and J. Rokach. 1988. Synthesis of ß-oxidation products as potential leukotriene metabolites and their detection in bile of anesthetized rat. Prostaglandins. 36: 291–302.[CrossRef][Medline]

  43. Audoly, L. P., B. Rocca, J. E. Fabre, B. H. Koller, D. Thomas, A. L. Loeb, T. M. Coffman, and G. A. FitzGerald. 2000. Cardiovascular responses to the isoprostanes iPF2{alpha}-III and iPE2-III are mediated via the thromboxane A2 receptor in vivo. Circulation. 101: 2833–2840.[Abstract/Free Full Text]

  44. Kunapuli, P., J. A. Lawson, J. Rokach, and G. A. FitzGerald. 1997. Functional characterization of the ocular prostaglandin F2{alpha} (PGF2{alpha}) receptor. Activation by the isoprostane, 12-iso-PGF2{alpha}. J. Biol. Chem. 272: 27147–27154.[Abstract/Free Full Text]

  45. Kunapuli, P., J. A. Lawson, J. Rokach, J. L. Meinkoth, and G. A. FitzGerald. 1998. Prostaglandin F2{alpha} (PGF2{alpha}) and the isoprostane, 8,12-iso-isoprostane F2{alpha}-III, induce cardiomyocyte hypertrophy. Differential activation of downstream signaling pathways. J. Biol. Chem. 273: 22442–22452.[Abstract/Free Full Text]

  46. Nourooz-Zadeh, J., E. H. C. Liu, B. Yhlen, E. E. Anggard, and B. Halliwell. 1999. F4-isoprostanes as specific marker of docosahexaenoic acid peroxidation in Alzheimer's Disease. J. Neurochem. 72: 734–740.[CrossRef][Medline]

  47. Praticò, D., C. M. Clark, V. M. Y. Lee, J. Q. Trojanowski, J. Rokach, and G. A. FitzGerald. 2000. Increased 8,12-iso-iPF2{alpha}-VI in Alzheimer's disease: correlation of a noninvasive index of lipid peroxidation with disease severity. Ann. Neurol. 48: 809–812.[CrossRef][Medline]


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M600327-JLR200v1
47/11/2515    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lawson, J. A.
Right arrow Articles by Rokach, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lawson, J. A.
Right arrow Articles by Rokach, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Journal of Biological Chemistry 
 Molecular and Cellular Proteomics   ASBMB Today 
Advertisement
spacer
Advertisement
Advertisement