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Resolvins D1, D2, and other mediators of self-limited resolution of inflammation in human blood following n-3 fatty acid supplementation.

Inflammation is a major mechanism involved in many human diseases. Resolution of inflammation is an active process regulated by biochemical mediators and receptor-signaling pathways and driven by proresolving mediators. Serhan et al. (1) first described these potent antiinflammatory and proresolving lipid mediators derived from polyunsaturated fatty acids. They include lipoxins derived from arachidonic acid, E-series resolvins derived from the long-chain n-3 fatty acid eicosapentaenoic acid (EPA) [2] (Fig. 1) and D-series resolvins protectins/neuroprotectins and maresins, all derived from the n-3 fatty acid docosahexaenoic acid (DHA) (Fig. 2) (2-5). These mediators, acting via G-coupled protein receptors (6), have potent antiinflammatory and proresolving actions (7, 8) and reduce airway inflammation (9,10), dermal inflammation (11), colitis (12), arthritis (13), and postoperative pain (14). Studies have shown that these mediators increase with time during the inflammatory process (1, 15). Two series of resolvins and protectins have been identified. One series includes those derived from EPA and DHA via lipoxygenase metabolism, referred to as the S-resolvins, S-protectins, and S-maresins (Figs. 1 and 2). The second series includes those derived from aspirin-triggered cyclooxygenase (COX-2) or cytochrome P450 metabolism of EPA and DHA. These lipid mediators are R-resolvins and R-protectins also known as aspirin-triggered resolvins/protectins (2, 3, 16, 17).



The aim of this study was to develop an assay that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously measure a number of lipid mediators of self-limited resolution of inflammation in human blood and to use this assay to measure their blood concentration following n-3 fatty acid supplementation. We also aimed to determine if the method of blood collection affects the measured concentration of lipid mediators.

Materials and Methods

18R/S-hydroxy-5Z,8Z,11Z,14Z,16E-eicosapentaenoic acid (18R/S-HEPE); 17S-hydroxy-4Z,7Z,10Z,13Z,15E, 19Z-docosahexaenoic acid (17S-HDHA); 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z- docosahexaenoic acid (RvD1 or Resolvin D1); 7S,8R,17R-trihydroxy 4Z,9E,11E,13Z,15E19Z-docosahexaenoic acid (17R RvD1 or 17R-Resolvin D1); 7S,16R,17S-trihydroxy4Z,8E,10Z,12E,14E,19Z- docosahexaenoic acid (RvD2 or Resolvin D2); 10S,17S-dihydroxy-4Z,7Z,11E,13Z,15E, 19Z-docosahexaenoic acid (10S,17S-diHDHA); and leukotriene [B.sub.4]-[d.sub.4] ([LTB.sub.4]-[d.sub.4]) were purchased from Cayman Chemicals. Bond ElutC18, 500 mg, 3-mLcartridgeswere from Agilent technolgies. Zorbax eclipse XDB C18 (2.1 X 100 mm X 3.5 [micro]m) columns were purchased from Agilent Technologies. All solvents were of HPLC grade. Water was obtained by using an Arium 611 VF water purification system (Sartorius Stedim Biotech). BD Vacutainer[R] SST[TM] II Advance tubes (3.5 mL), BD Vacutainer Citrate tubes (9NC, 0.0109 mol/L, 2.7 mL), and BD Vacutainer PST[TM] II tubes (lithium heparin 51 IU, 3 mL) were purchased from BD Vacutainer (Becton Dickinson). EDTA tubes (5 mL) contained dipotassium EDTA at a final concentration of 1 g/L blood. The 10R,17S-dihydroxy-4Z,7Z,11E,13E,15Z,19Z- docosahexaenoic acid (PD1 or protectin D1) standard was kindly provided as a gift by Professor Charles N. Serhan (Harvard Medical School, Boston, MA, USA).


Serum or plasma (1 mL) was thawed at room temperature and 500 pg [LTB.sub.4]-[d.sub.4] added as the internal standard. After dilution with 2 mL of100 mmol/L sodium acetate (pH 3) and acidification with acetic acid to pH 3, samples were applied to solid-phase extraction cartridges (Bond Elut C18 500 mg) and washed with 2 mL water and 2 mL hexane. Resolvins and protectins eluted with 2 mL ethyl acetate, dried under nitrogen and then reconstituted in 100 [micro]L of 5 mmol/L ammonium acetate (pH = 9)/ methanol (50/50; vol/vol) for analysis by LC-MS/MS.


The LC-MS/MS analysis was performed on a Thermo Scientific TSQ [Quantum.sup.Ultra] triple-quadrupole LC-MS system equipped with an electrospray ionization source (ESI) operated in the negative ion mode. Instrument control and data acquisition were performed with Xcalibur 2.0.7 software. LC was performed on a Zorbax Eclipse XDB C18 column at ambient temperature. The mobile phases were (A) 5 mmol/L ammonium acetate pH = 9, and (B) methanol at a flow rate of 400 [micro]L/min. Chromatography used solvent A/B (50/50, vol/vol) from 0 to 1min, then changed to A/B (30/70, vol/vol) from 1 to 15 min, changed to A/B (5/95, vol/vol) from 15 to 17min , and finally changed to A/B (50/50, vol/vol) from 18 to 21min. For optimization of the mass spectrometer, standards 18R/S-HEPE, 17S-HDHA, RvD1, 17R-RvD1, RvD2, 10S,17S-diHDHA, PD1, and [LTB.sub.4]-[d.sub.4] (internal standard) at a concentration of 10 ng/[micro]L were individually introduced into the mass spectrometer by direct infusion with a syringe pump at a flow rate of 10 [micro]L/min into the HPLC solvent flow (flow rate 0.2 mL/ min). The operating conditions for mass spectral analysis were as follows: spray voltage, 3500 V; capillary temperature and voltage, 350[degrees]C and 35 V, respectively; sheath gas (nitrogen) and auxiliary gas pressure 60 and 50 psi, respectively. The mass spectrometer was employed in MS/MS mode with argon used as the collision gas (1.2 mTorr).


Quantitative analysis was performed using calibration curves of standards prepared in plasma rendered free of proresolving lipid mediators (stripped plasma) by passage through a solid-phase extraction column (Bond Elut, C18 500 mg) as described above. Linearity was determined by assaying increasing amounts of each lipid mediator in duplicate (0, 25, 50, 100, 200, 500, 1000 pg/mL) and a fixed amount of the internal standard [LTB.sub.4] [d.sub.4] (500 pg). The limit of detection was calculated using a signal-to-noise ratio of 3. The limit of quantification was determined using a signal-to-noise of 10 as described by Masoodi et al. (18).


Analyte recovery was determined by adding mixtures of the standards in the concentration range (50, 500, and 1000 pg/mL, n = 15 replicates) to stripped plasma.


Venous blood (15 mL) was collected into EDTA from 10 healthy volunteers and kept on ice until centrifugation (1500g at 4[degrees]C). Pooled plasma was stripped as described above and 1-mL aliquots, stored at -80[degrees]C, were used to determine the intra- and interassay variation of the assay. Intraassay variation was calculated from analysis in triplicate of plasma spiked with 3 concentrations (50, 500, and 1000 pg/mL) of 18R/S-HEPE; 17S-HDHA; RvD1; 17R-RvD1; RvD2; 10S,17S-DiHDHA; and PD1. Interassay variation was calculated from triplicate analysis of plasma spiked with 3 concentrations (50, 500, and 1000 pg/mL) on 5 separate days. [LTB.sub.4]-[d.sub.4] (500 pg) was added before solid-phase extraction.



Blood was obtained from healthy volunteers recruited from the general population who were already enrolled in an n-3 fatty acid supplementation trial. There were 15 men and 5 women, aged 50-67 years [mean (SD) 59(5) years]. In this population the mean (SD) BMI was 28(5) kg/[m.sup.2], systolic/diastolic blood pressure was 118 (14)/71(6) mmHg, fasting cholesterol was 5.0 (0.8) mmol/L, triglycerides 1.3 (0.5) mmol/L, and glucose 5.0 (0.3) mmol/L. Plasma and serum samples were collected after 3 weeks of taking 4 g fish oil/day (Blackmores Omega Daily[R], 35% EPA and 25% DHA).

The participants gave informed written consent to participate in the study, which was approved by the human research ethics committee of the University of Western Australia. Fasting venous blood samples (15 mL) were prepared as serum (3.5 mL blood) or plasma from EDTA (5 mL blood), citrate (2.7 mL blood), or heparin (3 mL blood). Blood samples were collected on ice and centrifuged at 4[degrees]C, and the supernatant (plasma or serum) was stored at 80[degrees]C until analysis.


SPSS v19.0 was used for statistical analysis. Values are presented as means and SDs or SEs. The linear response each standard curve in stripped plasma was assessed using the coefficient of determination ([R.sup.2]).


With LC-MS/MS we showed that individual standards of lipid mediators were well-resolved chromatographically (Fig. 3). MS/MS used collision energy optimized for each standard to generate the most abundant product ions and monitored ions specific for each lipid mediator. The most prominent transition ions monitored in MS/MS for each lipid mediator (summarized in Fig. 3) were: RvD2, retention time ([R.sub.t]) = 3.31 min, m/z 375.2 [M-1] (molecular ion--1 mass unit), prominent product ions at m/z 174.9, 215, 259, 277, 295, and 357 with optimized collision energy 23, 18, 14, 14, 18, and 14 eV, respectively; RvD1 and 17R-RvD1, [R.sub.t] = 4.10 and 4.36 min, respectively, m/z 375.1 [M-1], prominent product ions at m/z 121, 135.1, 215.1, and 233.1 with optimized collision energy 31, 19, 18, and 15 eV, respectively; 10S,17S-DiHDHA and PD1, [R.sub.t] = 7.58 and 7.95 min, respectively, m/z 359.2 [M-1], prominent product ions at m/z 137, 153, 188.2, and 206.2 with optimized collision energy 22, 17, 22, and 18 eV, respectively; [LTB.sub.4]-[d.sub.4], [R.sub.t] = 8.23 min, m/z339.2 [M-1]; prominent product ion at m/z 197 with optimized collision energy 18 eV; 18R/S-HEPE, [R.sub.t] = 10.50 min, m/z 317.2 [M-1]; prominent product ions at m/z 215.1, 255.1, 259.1, 273, and 299.2 with optimized collision energy 15, 15, 14, 13, and 13 eV, respectively; 17SHDHA, [R.sub.t] = 14.00 min, m/z 343.1 [M-1]; prominent product ions at m/z 201.1, 227.1, 245.1, 281.1, and 325.2 with optimized collision energy 14, 18, 13, 14, and 12 eV, respectively.

Calibration curves prepared from standard solutions showed the assay had excellent linearity up to 1000 pg (Fig. 4). The coefficient of determination (.R2) for each was [greater than or equal to] 0.97. The limit of detection was 3 pg on-column and the limit of quantification was 6 pg on-column. The mean recovery of each lipid mediator in stripped plasma spiked with 50, 500, or 1000 pg of standards was 73%, 96%, and 99%, respectively. The within-day precision of the assay was 1.3%-13.5% in samples analyzed in triplicate over a concentration range of 50-1000 pg/mL (Table 1). The between-day precision over the same concentration range performed in triplicate on 5 separate days ranged from 8.2% to 14.9% (Table 1).

RvD2, RvD1, 17R-RvD1, 18R/S-HEPE, and 17R/ S-HDHA were measured in serum and plasma from healthy volunteers who had been enrolled in an n-3 fatty acid supplementation trial for 3 weeks (Table 2). The concentration of 18R/S-HEPE and 17R/S-HDHA was approximately 5-10-fold greater than that of their downstream pathway products. In plasma, both 18R/ S-HEPE and 17R/S-HDHA were approximately 2-fold greater than in serum. Serum concentrations ofRvD2, RvD1, and 7S,8R,17R-trihydroxy-4Z,9E,11E,13Z, 15E19Z-docosahexaenoic acid (17R-RvD1) were comparable to those in plasma collected into different anticoagulants. There were no significant differences for any of the measured lipid mediators between plasma collected into the different anticoagulants. 10S,17S-DiHDHA and PD1 in plasma and serum were below the limit of quantification.



We describe for the first time an assay that simultaneously measures a number of EPA- and DHA-derived resolvins and protectins using LC-MS/MS in human samples following n-3 fatty acid supplementation. The assay has excellent reproducibility and precision. To date the reports on resolvins and protectins have mainly focused on in vitro studies or small animal models of acute inflammation and there is a paucity of data on the concentrations of these lipid mediators in humans. We have shown that following n-3 fatty acid supplementation, the pathway precursors 18R/S-HEPE and 17R/S-HDHA, as well as the resolvins RvD2, RvD1, and 17R-RvD1, were present in concentrations that are known to have potent antiinflammatory and proresolving effects. Plasma concentrations of 18R/SHEPE and 17R/S-HDHA, but not measured resolvins, were approximately 2-fold greater than in serum.

We achieved optimization for the measurement of resolvins and protectins by LC-MS/MS. Using 5 different stripped plasma samples we determined that there were no interfering peaks present in the biological matrix at the retention times corresponding to any of the resolvins and protectins measured. The individual standards were well resolved chromatographically. In analyses we selected the most prominent product ion for quantification and a second ion to confirm identity. The assay had detection and quantification limits of 3 pg and 6 pg on-column, respectively, and showed linearity up to 1000 pg/mL with excellent recovery and precision.

In our assay the enantiomers RvD1 and 17R-RvD1 were separated chromatographically with retention times 4.10 and 4.36 min respectively. Baseline resolution of these two lipid mediators can be achieved using a chiral liquid chromatography column as reported by Oh et al. (19). Given 18R- and 18S-series 5S, 12R, 18R-trihydroxy-6Z, 8E, 10E, 14Z, 16E-eicosapentaenoic acid (RvE1) and 5S, 18R-dihydroxy-6E, 8Z, 11Z, 14Z, 16E-eicosapentaenoic acid (RvE2) are highly labile lipid mediators, we used racemic 18R/S-HEPE, which represents the precursor of these resolvins derived from EPA metabolism via lipoxygenase, COX-2, or P450 (Fig. 1). Thus measurement of 18R/S-HEPE may represent total EPA-derived resolvin capacity. The enantionmers 18S-HEPE and 18R-HEPE can be resolved with baseline resolution by using chiral chromatography (19). Our analyses showed that 17S-HDHA and 17R-HDHA eluted as a single peak on LCMSMS and had the same fragmentation pattern (data not shown). Although the 17S-HDHA was used to construct calibration curves and establish reproducibility, plasma and serum samples may contain both 17S-HDHA and 17R-HDHA. Measurement of 17R/S-HDHA most likely represents total DHA-derived resolvin capacity.

In healthy volunteers who took 4 g daily fish oil, which provided approximately 1.4 g/day EPA and 1.0 g/day DHA, for 3 weeks, we detected measurable concentrations of RvD2, RvD1, 17R-RvD1, 18R/S-HEPE, and 17R/S-HDHA in 1 mL of serum or plasma. The biological actions of RvD2, RvD1, and 17R-RvD1 have been extensively studied and each has been shown to have potent antiinflammatory and proresolving actions in in vitro and in vivo small animal studies (1). 18R/S-HEPE is the precursor of 18R- and 18S-series RvE1 and RvE2. 18R-HEPE was first identified in inflammatory exudates from mice treated with n-3 fatty acids and aspirin (20). Oh et al.(19) also showed that both 18R-HEPE and 18S-HEPE were present in the serum of health volunteers 3 h after a single dose of 1g EPA. Volunteers had previously taken 81 mg aspirin at 12 and 24 h before consuming EPA. We have shown that 3 weeks of n-3 fatty acid supplementation led to serum concentrations of 18R/S-HEPE of 191 pg/mL that compares favorably with the total 18R-HEPE and 18S-HEPE concentration (122 pg/mL) reported by Oh et al. (19). 17R/S-HDHAare the precursors of the 17Rand 17S-series of RvD1 and RvD2 (Fig. 2). To our knowledge this is the first report of the presence of 17R/S-HDHA, and RvD1 and RvD2 in human blood following oral supplementation with n-3 fatty acids.

Proresolving lipid mediators are locally acting autocoids whose concentrations would be expected to be highest at sites of inflammation where they exert their effects. An important finding of our study is that the concentrations of the potent antiinflammatory and proresolving agents RvD1 and RvD2 detected in blood from healthy humans after n-3 fatty acid supplementation for 3 weeks are within the bioactive range observed in isolated human leukocytes (21, 22) and in in vivo studies in mice (12, 23).

Previous reports have used plasma or serum for measurement of resolvins and protectins and yet to our knowledge this important aspect has not been examined. We have shown that in human blood the concentration of 17R/S-HDHA was comparable to that of 18R/S-HEPE and both were present in quantities at least 5-10-fold greater than that of their downstream pathway products. The concentration of both 18R/SHEPE and 17R/S-HDHA in plasma, regardless of anticoagulant, was approximately 2-fold greater than in serum. In contrast, serum concentrations of RvD2, RvD1, and 17R-RvD1 were comparable to those in plasma collected into different anticoagulants. The type of anticoagulant used for blood collection did not affect the concentration of the lipid mediators. The lower 18R/S-HEPE and 17R/S-HDHA in serum suggests that degradation of these lipid mediators may be occurring during the clotting process. Therefore, plasma concentrations may be more representative of circulating 18R/S-HEPE and 17R/S-HDHA.

Following n-3 fatty acid supplementation we observed peaks corresponding with 10S,17SDiHDHA and PD1 in plasma and serum but these were below our criteria for the limit of quantification. It is possible that extraction of larger volumes of plasma or serum could enable their detection. However, to our knowledge 10S, 17S-DiHDHA, and PD1 have not been reported in human blood. 10S,17S-DiHDHA has been measured in murine peritonitis but is produced only in trace quantities by human neutrophils (24). PD1 has potent immunoregulatory and neuroprotective actions (25) and is generated by isolated human leukocytes and tissues (24) and in particular by neural tissues where it is referred to as NPD1 (25). It is antifibrotic in the kidney and promotes wound-healing capacity (8).

The relevance of our findings to clinical medicine is highlighted by the range of conditions that may be influenced by RvD1 and RvD2. RvD1 has been shown to have antiinflammatory actions in animal models of inflammation. RvD1 provides protection against renal injury after ischemia reperfusion (26) and prevents neutrophil recruitment in peritonitis (27) and the dorsal skin air pouch (22). 17R-RvD1 is effective in preventing experimental colitis in the mouse (12). RvD2 is a potent regulator of leukocytes. It controls microbial sepsis by reducing levels of proinflammatory cytokines (28) and prevents experimentally induced colitis in mice (12).

RvD1 and RvD2 have both been implicated as antinociceptive agents. RvD1 reduces inflammatory and postoperative pain in rodent models (29, 30), has been shown to inhibit transient receptor potential ankyryn 1 (TRPA1) in the dorsal root ganglion (31), and is an effective antihyperalgesic agent in a rat model of adjuvant-induced arthritis (13, 32). In mice, RvD2 is a potent inhibitor of TRPV1 (transient receptor potential subtype vanilloid) and TRPA1, 2 types of TRP channels that are strongly implicated in the genesis of inflammatory pain. RvD2 blocks synaptic plasticity that contributes to the development and maintenance of inflammation-induced pain (31). Therefore, the discovery of physiologically relevant levels of these mediators in human blood after n-3 fatty acid supplementation has the potential to broaden our understanding of a range of mechanisms involved in different clinical conditions.

In conclusion, we have described for the first time an assay that uses LC-MS/MS to measure a number of potent mediators of self-limited resolution of inflammation in human blood with excellent reproducibility and precision. We have shown that the EPA- and DHA-derived pathway precursors, as well as several resolvins, are present in plasma and serum following n-3 fatty acid supplementation. These findings highlight that biologically relevant concentrations of mediators of self-limited resolution of inflammation are achievable in healthy humans with n-3 fatty acid supplementation and thereby contribute to a better understanding of the mechanisms by which n-3 fatty acids exert their antiinflammatory action.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: K.D. Croft, National Heart Foundation of Australia; A.E. Barden, National Heart Foundation of Australia; T.A. Mori, National Health and Medical Research Council of Australia, National Heart Foundation of Australia.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We acknowledge Lynette McCahon and Rachel Hendry for technical assistance.


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Emilie Mas, [1] * Kevin D. Croft, [1] Paul Zahra, [1] Anne Barden, [1] ([dagger]) and Trevor A. Mori [1] ([dagger])

[1] School of Medicine and Pharmacology, Royal Perth Hospital Unit, University of Western Australia, Perth, Australia.

[2] Nonstandard abbreviations: EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; COX-2, aspirin-triggered cyclooxygenase; LC-MS/MS, liquid chromatography-tandem mass spectrometry; 18R/S-HEPE, 18R/S- hydroxy5Z,8Z,11Z,14Z,16E-eicosapentaenoic acid; 17S-HDHA, 17S-hydroxy-4Z,7Z,10Z, 13Z,15E, 19Z-docosahexaenoic acid; RvD1, 7S,8R,17S-trihydroxy-4Z,9E, 11E,13Z,15E,19Z-docosahexaenoic acid; 17R-RvD1, 7S,8R,17R- trihydroxy4Z,9E,11E,13Z,15E19Z-docosahexaenoic acid; RvD2, 7S,16R,17S-trihydroxy 4Z,8E,10Z,12E,14E,19Z-docosahexaenoic acid; 10S,17S-diHDHA, 10S,17S dihydroxy-4Z,7Z,11E,13Z,15E,19Z-docosahexaenoic acid; [LTB.sub.4]-[d.sub.4] leukotriene [B.sub.4]-[d.sub.4]; PD1, 10R,17S-dihydroxy-4Z,7Z,11E,13E,15Z,19Z- docosahexaenoic acid; Rt, retention time; [M-1], molecular ion--1 mass unit; RvE1, 5S,12R,18R- trihydroxy-6Z,8E,10E,14Z,16E- eicosapentaenoic acid; RvE2, 5S,18R-dihydroxy6E,8Z,11Z,14Z,16E- eicosapentaenoic acid; TRPA1, transient receptor potential ankyryn 1.

* Address correspondence to this author at: School of Medicine and Pharmacology, University of Western Australia, Medical Research Foundation Bldg., GPO Box X2213, Perth, Western Australia 6847. Fax +61-8-9224-0246, e-mail

([dagger]) Anne Barden and Trevor A. Mori contributed equally to the work, and both should be considered as first authors.

Received May 22, 2012; accepted July 27, 2012.

Previously published online at DOI: 10.1373/clinchem.2012.190199
Table 1. Within- and between-day assay variation of resolvins
and protectins in human stripped plasma.

Concentration       RvD1       RvD2     17R-RvD1   10S,17S-DiHDHA

Within day
  50 pg/mL
    Mean             48.2       54.2       40.4          60.4
    SD                1.8        1.7        0.9           3.0
    RSD (a) (%)       3.7        3.1        2.2           5.0
  500 pg/mL
    Mean            392.4      533.8      521.0         550.6
    SD               14.8       22.0       37.3          13.9
    RSD (%)           3.8        4.1        7.2           2.5
  1000 pg/mL
    Mean            886.2     1256.2      938.2        1317.3
    SD               30.7       65.6       61.5          81.1
    RSD (%)           3.5        5.2        6.6           6.2
Between day
  50 pg/mL
    Mean             34.6       34.0       28.8          42.1
    SD                4.6        4.7        3.4           5.8
    RSD (%)          13.3       13.8       11.9          13.8
  500 pg/mL
    Mean            444.6      547.6      439.6         603.5
    SD               64.9       58.5       66.5          61.1
    RSD (%)          14.6       10.7       15.1          10.1
  1000 pg/mL
    Mean           1042.5     1352.5      964.1        1281.0
    SD              155.3      152.9       98.8         108.9
    RSD (%)          14.9       11.3       10.2           8.5

Concentration       PD1     18R/S-HEPE   17S-HDHA

Within day
  50 pg/mL
    Mean            33.4       50.9        32.7
    SD               4.5        2.6         0.4
    RSD (a) (%)     13.5        5.1         1.3
  500 pg/mL
    Mean           559.3      417.9       311.8
    SD              14.9       13.9        28.5
    RSD (%)          2.7        3.3         9.2
  1000 pg/mL
    Mean          1360.0      813.2       558.7
    SD              61.4       37.0        40.3
    RSD (%)          4.5        4.6         7.2
Between day
  50 pg/mL
    Mean            26.7       50.6        29.9
    SD               3.0        6.3         4.3
    RSD (%)         11.3       12.4        14.5
  500 pg/mL
    Mean           592.2      424.0       270.3
    SD              55.4       42.9        40.4
    RSD (%)          9.4       10.1        14.9
  1000 pg/mL
    Mean          1316.9      919.1       556.6
    SD             108.5       94.3        66.1
    RSD (%)          8.2       10.3        11.9

(a) RSD, relative SD.

Table 2. Mean concentration (pg/mL) of lipid mediators in human
blor plasma collected in EDTA, heparin, or citrate following n-3
fatty acid supplementation. (a)

Lipid mediator,
     pg/mL            Serum           EDTA           Heparin

RvD1               24.4 (2.5)       31.4(4.6)      33.0 (4.0)
RvD2               26.6 (4.7)      26.4 (3.6)      29.9 (3.8)
17R-RvD1           55.3 (6.0)      60.8 (7.3)      73.8 (7.4)
10S,17S DiHDHA      <LOQ (b)          <LOQ            <LOQ
PD1                   <LOQ            <LOQ            <LOQ
18R/S-HEPE         190.8(16.6)    385.7 (52.6)     310.0(22.8)
17R/S-HDHA        175.3 (32.2)    364.7 (65.0)     319.6(64.5)

Lipid mediator,
     pg/mL           Citrate

RvD1               40.6 (7.3)
RvD2               32.1 (4.9)
17R-RvD1           70.2 (4.5)
10S,17S DiHDHA        <LOQ
PD1                   <LOQ
18R/S-HEPE        367.8 (28.0)
17R/S-HDHA        486.2 (227.3)

(a) Values are given as mean (SE)..

(b) <LOQ = concentration below criteria for the limit of
quantification (25pg) in 1 mL of serum or plasma.
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Article Details
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Title Annotation:Lipids, Lipoproteins, and Cardiovascular Risk Factors
Author:Mas, Emilie; Croft, Kevin D.; Zahra, Paul; Barden, Anne; Mori, Trevor A.
Publication:Clinical Chemistry
Article Type:Report
Geographic Code:8AUST
Date:Oct 1, 2012
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