Arm et al. Lipids in Health and Disease 2013, 12:141 Impact of botanical oils on polyunsaturated fattyacid metabolism and leukotriene generation inmild asthmatics Jonathan P Arm1,4,5,9, Joshua A Boyce4,5,7, Lin Wang1, Heng Chhay1, Muhammad Zahid2, Vaishali Patil2,Usha Govindarajulu1, Priscilla Ivester6,7, Kelly L Weaver6,10, Susan Sergeant7,8, Elliot Israel2,3,5 and Floyd H Chilton6,7* Background: Dietary supplementation with botanical oils that contain n-6 and n-3 eighteen carbon chain (18C)-PUFAsuch as γ linolenic acid (GLA, 18:3n-6), stearidonic acid (SDA, 18:4n-3) and α linolenic acid (ALA, 18:3n-3) have beenshown to impact PUFA metabolism, alter inflammatory processes including arachidonic acid (AA) metabolism andimprove inflammatory disorders.
Methods: The diet of mild asthmatics patients was supplemented for three weeks with varying doses of two botanicalseed oils (borage oil [Borago officinalis, BO] and echium seed oil [Echium plantagineum; EO]) that contain SDA, ALA andGLA. A three week wash out period followed. The impact of these dietary manipulations was evaluated for severalbiochemical endpoints, including in vivo PUFA metabolism and ex vivo leukotriene generation from stimulatedleukocytes.
Results: Supplementation with several EO/BO combinations increased circulating 20–22 carbon (20–22C) PUFAs,including eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and dihommo-gammalinolenic acid (DGLA),which have been shown to inhibit AA metabolism and inflammation without impacting circulating AA levels. BO/EOcombinations also inhibited ex vivo leukotriene generation with some combinations attenuating cysteinyl leukotrienegeneration in stimulated basophils by >50% and in stimulated neutrophils by >35%.
Conclusions: This study shows that dietary supplementation with BO/EO alters 20–22C PUFA levels and attenuatesleukotriene production in a manner consistent with a reduction in inflammation.
Keywords: Asthma, Gammalinolenic acid, Stearidonic acid, Inflammation, Leukotrienes, Borage oil, Echium oil effects, which include contraction of bronchial smooth Asthma is a complex disease which involves smooth muscle, vasodilatation, and mucus secretion within the muscle contraction and inflammation that result in airways [Additionally, LTB4 is a potent chemo- narrowing and obstruction of the airway. Arachidonic attractant for granulocytes, effector T cells, and mono- acid (AA) metabolism via the 5-lipoxygenease pathway cytes, acting at a specific GPCR, BLT1 Collectively, to form leukotrienes has been demonstrated to be par- leukotrienes have numerous proinflammatory properties, ticularly important to the pathology of asthma. Specific- and leukotriene modifying drugs have proven effective in ally, the cysteinyl leukotrienes, LTC4, LTD4, and LTE4, the management of asthma and allergic rhinitis act at GPCRs, CysLT1R and CysLT2R, to elicit their In addition to pharmacologic modifiers, supplementa- tion of diets with fatty acid-based marine and botanical oil * Correspondence: supplements have been demonstrated to reduce the sever- 6Department of Physiology/Pharmacology, Wake Forest School of Medicine, ity of several inflammatory disorders including asthma.
Medical Center Blvd, 27157, Winston-Salem, NC, USA7 One of the primary mechanisms thought to be responsible Center for Botanical Lipids and Inflammatory Disease Prevention, Wake Forest School of Medicine, Medical Center Blvd, 27157, Winston-Salem, NC, for their efficacy has been the capacity of n-6 and n-3 polyunsaturated fatty acids to alter AA metabolism to Full list of author information is available at the end of the article 2013 Arm et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

Arm et al. Lipids in Health and Disease 2013, 12:141 form eicosanoids. For example, dietary supplementation It has been long appreciated that natural products may with fish oils that contain eicosapentaenoic acid (EPA; contain a complex mixture of several active ingredients 20:5, n-3) inhibits leukotriene generation, likely through with synergistic biological effects. As discussed above, bo- substrate competition of EPA with AA for the action of tanical seed oils contain several 18C-PUFAs that have the cytosolic phospholipase A2 and 5-lipoxygenase .
potential to impact diseases that are driven by eicosanoid In regard to botanical oils, borage seed oils contain the generation. Several studies have examined the impact of 18 carbon PUFA, gammalinolenic acid (GLA; 18:3, n-6) providing individual botanical oils that contain a high pro- that is elongated by most tissues and inflammatory cells to portion of their total fatty acids as a putative PUFA active dihommo-gammalinolenic acid (DGLA) (Figure Newly- ingredient; however, little is known regarding the bio- formed DGLA is then incorporated into membrane phos- chemical interactions of adding more than one botanical pholipids. Like EPA, DGLA has the capacity to compete oil containing several potential PUFA active ingredients.
with AA for eicosanoid production. DGLA is also The primary objective of this current study was to exam- converted to prostaglandin (PG)H1 via cyclooxygenase en- ine the biochemical impact of adding PUFAs found in two zyme(s), which is then converted to PGE1. PGE1 has been botanical seed oils on PUFA metabolism and leukotriene illustrated to have anti-inflammatory effect in both animals generation in mild asthmatic patients.
In addition, seed oil from another member of the Materials and methods Boraginaceae family, Echium plantagineum contains both n-6 and n-3 18C-PUFAs, including a GLA, α-linolenic acid Thirty-seven asthmatic subjects (ages 18–64) were (ALA; 18:3, n-3) and stearidonic acid (SDA; 18:4, n-3). The recruited. Written informed consent was obtained from conversion of ALA to EPA and DHA is poor in humans all subjects prior to enrollment. Asthma was diagnosed by which is believed to be a result of the inefficiency of the presence of variable airflow obstruction or by a history the initial rate-limiting step (Δ-6 desaturase, FADS2 gene) of treatment for asthma with documented airways hyper- involved in 20–22 carbon PUFA biosynthesis. However responsiveness to methacholine []. Histories (medical SDA is downstream of Δ-6 desaturase and is 4 to 5-fold and respiratory), a brief physical examination and routine more efficiently converted to EPA than ALA clinical test were used to exclude the presence of signifi- Additionally, SDA has been demonstrated to block in vitro cant co-morbid diseases. The inclusion criteria were: 1) leukotriene generation and in vivo inflammatory processes male or female 18 years to 65 years of age; 2) asthma with FEV1 50 to 90% of predicted, or personal best; and 3) ω-3 series
ω-6 series
α-linolenic acid 18:3
Stearidonic acid 18:4
γ-linolenic acid
ω -3 Arachidonic acid 20:4
Dihomo- -
γ linolenic acid
Eicosapentaenoic acid 20:5
Arachidonic acid
Docosapentaenoic acid 22:5
Docosahexaenoic acid 22:6
Figure 1 Pathways for metabolism of n-6 (left) and n-3 (right) PUFAs in humans. The pathway depicts the synthesis of 20 and 22 carbonPUFAs from the essential, dietary PUFAs, α-linolenic acid (n-3) and linoleic acid (n-6). The PUFAs derived from borage oil (linoleic and gamma-linolenic acids, both n-6) and echium (α-linolenic, n-3; stearidonic, n-3; linoleic and gamma-linolenic acids, both n-6) would be expected to enterthe pathways at the indicated points.
Arm et al. Lipids in Health and Disease 2013, 12:141 improvement in FEV1 > 12% after administration of a for three months prior to entry into the study no subject beta-2 agonist. The exclusion criteria were: 1) pregnant or used omalizumab, a monoclonal antibody against IgE.
nursing; 2) smoking history of > 10 pack years or active The study was approved by the Partners Human Sub- smoking within the previous year; 3) use of asthma treat- jects Research Committee. An investigator-initiated IND ments that potentially alter leukotriene biosynthesis includ- was obtained from the Food and Drug Administration ing theophylline and oral steroids; 4) dietary supplements (IND number 74,110). Because the outcomes in this with fatty acids or other products that may interfere with study were entirely biochemical with no assessment leukotriene generation; 5) treatment within the previous of clinical efficacy, the study was not registered at three months with omalizumab (monoclonal antibody di- rected against IgE); 6) use of non-steroidal anti-inflammatory drugs in the week prior to any measurements Dietary fatty acids of ex vivo leukotriene generation; 7) a history of aspirin- Capsules containing echium seed oil (1000 mg) or borage sensitive asthma; 8) significant abnormalities in CBC, differ- seed oil (1300 mg) were supplied by Bioriginal (Saskatoon, ential white cell count, renal function, and liver function, or SA, Canada). The fatty acid contents of the oils were vali- urinalysis; and 9) any serious co-morbid medical condition.
dated by gas chromatography (Table Since the leaves of Subjects meeting the entry criteria were randomized Echium plantagineum contain pyrrolizidine alkaloid, an in- to one of the four study groups based on botanical oil dependent assay of this toxin was obtained, assessed by dosing (Table Subjects used rescue albuterol less than high performance TLC, and found it to be less than 4 ng/g twice a week, had nocturnal asthma less than twice a of oil (Chemisches Laboratorium Dr. Hermann Ulex Nachf, month, and had a forced expiratory volume in one sec- ond ≥75% predicted. During the month prior to entryinto the study, no subject used leukotriene modifiers, oral or high dose inhaled steroids, or theophylline, and The study goal was to test combinations of Boraginaceaefamily seed oils derived from Borago officinalis (BO) and Table 1 Study group compositions and daily dosing Echium plantagineum L. (EO) for their capacity to impact scheme for borage and echium seed oils in the four circulating PUFA levels and inhibit ex vivo leukotriene groups of subjects generation. After baseline determinations of plasma fatty acid levels and ex vivo leukotriene generation, subjects were randomized to dosing groups (Table ingesting a Table 2 Fatty acid profile of botanical oils consumed by asthmatic subjects No. of subjects withdrawn % of Total Fatty Acids Echium Oil Dose (g) No. of daily capsules Borage Oil Dose (g) No. of daily capsules No. of daily capsules The number of daily capsules, containing 1 g (EO) or 1.3 g (BO) oil per capsule, and the amount of ALA, SDA and GLA provided by the encapsulated echiumand borage oils is shown for each dosing group. Recruitment into Group 1 was stopped after the interim analyses showed that the highest dose of EO was not required to prevent elevations of AA levels in circulating plasma.
Arm et al. Lipids in Health and Disease 2013, 12:141 constant dose of GLA ( 1.5 g daily), 0.25 g to 1.75 g SDA constipation, loose stools, and/or abdominal pain or dis- and 0.57 g to 4.02 g ALA in divided doses (three times a comfort. With the exception of the 3 subjects who with- day) for 3 weeks.
drew due to gastrointestinal discomfort (Groups 2, 3 and Aside from the addition of supplements, the subject's 4), these symptoms were mild and transient, occurred in diets were not altered before or during the study. The the first few days of the study and resolved within 2 to ‘typical' western diet provides very small quantities of 3 days while still taking BO/EO combinations. Other ad- GLA and SDA, and it is difficult to measure these PUFA verse events included cold-like symptoms (n = 7: Group 1 in circulating or cellular lipids. GLA and SDA are typic- ally only observed when individuals are consuming a ryngitis (n = 2; one each in Groups 2 and 4), mild symp- GLA- or SDA- containing supplement such as borage, toms of asthma associated with seasonal allergies (n = 2, evening primrose, black currant, or echium, and this was an one each in Groups 3 and 4), transient wrist pain (n = 1; exclusion criterion of the study. ALA (n-3) makes up 1% group 3), and transient itchy rash (n = 1; Group 4). Two of the typical western diet and so the supplement may have other subjects withdrew from the study due to the develop- some impact on dietary levels. Approximately 7% of energy ment of itch and mild asthma flare (Group 4) and unfore- in the western diet is LA, and thus the quantities of seen extension of an out-of-town business trip (Group 1).
LA in the supplements would have little total impact There were no significant changes in vital signs in any on overall linoleic acid (n-6) consumption.
study subjects.
Concentrations of GLA provided to human subjects as There were no consistent or significant changes in bio- borage oil alone have been shown to induce increases in chemical laboratory parameters or urinalysis during the circulating AA levels []. It was our hypothesis that the study (data not shown). However, in four of the individ- presence of the n-3 18C-PUFA SDA in echium oil would uals who entered the study (2 male, 2 female) there was a prevent such an increase. However, to assure that there decrease in circulating hemoglobin of >1.0 g/dl. Across all were no elevations of circulating AA out of the normal study groups there was on average a decrease in circulat- range, an interim analysis was carried out during the early ing hemoglobin of 0.45 g/dl (13.8 to 13.3 g/dl; p < 0.001), phase of the study. The interim analyses revealed that AA which did not vary significantly between study groups levels remained constant in all groups and consequently, (p = 0.09 for differences among groups across time). In the study was then completed. Blood and urine were col- three individuals, the value dipped just below the range of lected weekly for assessment of hematological indices, normal. There was an accompanying decrease in red cell liver and renal function, plasma fatty acids, ex vivo leuko- count from 4.58 to 4.39x1012 /liter with no change in triene generation. After three weeks of supplementation, mean corpuscular volume. A decrease in hemoglobin subjects entered a 3 week wash-out phase, in which concentration was not seen in all subjects. There were plasma fatty acids and ex vivo leukotriene generation were no accompanying decreases in white cell count or measured weekly. After dropouts, 6 to 11 individuals com- platelet count.
pleted each arm of the study.
Subjects reported any change in their medical condi- Plasma and botanical oil fatty acids analysis tion at each study visit and used diary cards to record all Fatty acid methyl esters were prepared in duplicate medical symptoms and to log intake of study oils. Com- plasma samples (100 μl) following a modification of pliance was monitored by medication diaries and counts Metcalfe et al. and analyzed by gas chromatography of returned capsules. These assessments indicated at as previously described [Encapsulated oils were suit- least 90% compliance by all subjects and plasma fatty ably diluted in hexane and submitted to fatty acid ana- acids measurements, in which no outliers were seen, also lysis in a manner comparable to that for plasma. Fatty reflected in a comparable adherence to the supplementa- acids in sample were identified based on retention times tion. As the end points of the study were entirely bio- of authentic fatty acid methyl esters (Supleco, Bellefonte, chemical, the subjects were not blinded (i.e. subjects PA, USA; Cayman Chemicals, Ann Arbor MI, USA; took different numbers of capsules depending on the Matreya, Pleasant Gap PA, USA; NuChek Prep, Elysian, group to which they were assigned). The objective of this biochemical study was to determine the impact of po-tentially bioactive PUFAs in combinations of BO and EO Ex vivo leukotriene generation on plasma fatty acid levels and ex vivo leukotriene pro- FcεRI-dependent generation of cysteinyl leukotrienes from duction. All individuals performing the biochemical as- basophils. Peripheral blood mononuclear cells (PBMC), says were blinded to the subjects' study group.
typically containing 3 to 4% basophils, were isolated The most common adverse events associated with taking from 20 mL of blood by density gradient centrifugation BO/EO combinations were gastrointestinal symptoms, oc- over Percoll (GE Healthcare, Piscataway, NJ) as de- curring in thirteen subjects, variously reported as gas, scribed ]. As previously described basophils Arm et al. Lipids in Health and Disease 2013, 12:141 were stimulated in PBMC by cross-linking of the high (p < 0.05, Figure white bar) than baseline in Group 1 affinity Fc receptor for IgE (FcεRI). Under these circum- (wk 3) and Group 3 (wk 2). SDA (18:4n-3) was not detect- stances, the basophil is the predominant, if not the only, able in the plasma at baseline but rose significantly during source of cysteinyl leukotrienes. Briefly, cells were supplementation with BO and EO (p < 0.0001, Figure primed with 10 μg/L IL-3 on ice and stimulated with black bar). As expected, the increase in plasma SDA varied 0.01 to 1.0 μg/mL of 15A5, an activating monoclonal between groups and was most marked in Group 1 in antibody to FcεRI, for 30 min at 37°C. Cysteinyl leukotri- which subjects consumed the largest dose of echium oil enes in the supernatant fluids were stored at −80°C until (p = 0.004 for differences among groups across time). In assayed by immunoassay (Cayman Chemical Company, each group, the plasma levels of EPA (20:5n-3; Figure Ann Arbor, MI or GE Healthcare) for LTC4 with 100% gray bar) and its elongation product DPA (22:5n-3; cross-reactivity with LTC5, 48% and 46% cross-reactivity Figure cross-hatched bar) rose significantly (p < 0.0001 with LTD4 and LTD5, respectively, and <10% cross- for each fatty acid) in a manner that was dependent on reactivity with LTE4 and LTE5.
the dose of echium oil ingested (p = 0.004 for group by A23187-stimulated generation of leukotrienes from neu- time interactions for each fatty acid). However, there was trophils. Neutrophils were isolated from heparinized blood no rise in circulating plasma DHA (22:6n-3; Figure by dextran sedimentation, density gradient centrifugation white striped bar) in any group (p = 0.14). During the through Ficoll Paque (GE Healthcare, Uppsala, Sweden), washout phase, plasma SDA, EPA and DPA rapidly and hypotonic lysis of contaminating red cells []. Neu- returned to baseline levels.
trophils (2 × 106) were stimulated (5 min, 37°C) with 0.1 With respect to n-6 fatty acids, plasma concentrations to 10 μmol/L A23187 (Sigma-Aldrich, St Louis, MO) in of GLA (18:3n-6; Figure black bar) and DGLA (20:3n-6; Hanks buffered salt solution containing 1.25 mmol/L cal- Figure white bar) rose in each group (p < 0.0001 for cium and magnesium salts, 25 mmol/L Hepes, and 1 g/L each fatty acid) with no significant difference between fatty acid-free bovine serum albumin. Leukotrienes in the groups (p = 0.06 and p = 0.63, respectively, for differences supernatants were resolved by reverse-phase HPLC (RP- among groups across time). Importantly, concentrations HPLC) as described and quantified by absorbance of plasma AA (20:4n-6; Figure , gray bar) remained con- at 280 nm (LTB4 and all-trans LTB4) and 235 nm (5- stant throughout with no significant change during sup- hydroxyeicosatetraenoic acid, 5-HETE).
plementation with BO and EO in any group (p = 0.39 foreffect of time, and p = 0.87 for group by time interactions).
Statistical analysis During the washout phase, plasma GLA and DGLA rap- Statistical analyses were performed using SAS Version idly returned to baseline levels.
9.2. We employed a repeated measures mixed model(rmm) analysis for each fatty acid to predict the response Ex vivo leukotriene generation in which repeated measures were taken on each subject Figure illustrates the effect of dietary supplementation at each visit (time) and the group and time interactions with a constant dose of BO and variable doses of EO on were modeled along with the main effects. The model ex vivo cysteinyl leukotriene generation from basophils utilized a compound symmetry correlation structure stimulated through FcεRI. Basophils are the only cell since the correlation between pairs of times would be among peripheral blood mononuclear cells that express similar and not expected to change across time. For ana- the fully functional heterotetrameric αβγ2 form of FcεRI lyses of ex vivo leukotriene generation by neutrophils and are the principal, if not the only, source of cysteinyl and basophils, we also considered the effect of a range leukotrienes when this population of cells is stimulated of doses of each stimulus at each visit within the same through this receptor []. We therefore stimulated baso- modeling. We then used adjusted least square mean dif- phils at 3 to 4% purity in PBMC with an activating anti- ferences obtained from each rmm model to compare the body to FcεRI, before (baseline) and at weekly intervals for differences between groups and between times within a three weeks while subjects supplemented their diet with single group. All analyses were conducted at a 0.05 level borage and echium seed oils. Because of the marked dif- of significance.
ference among individuals in absolute quantities ofcysteinyl leukotrienes generated by basophils, varying from 1 to 90 ng/106 basophils, data are expressed as a per- Plasma fatty acids centage of maximal leukotriene generation per million ba- The effects of dietary supplementation with BO and EO sophils prior to treatment with BO and EO. Compared to on n-3 and n-6 PUFA levels are provided in Figure and baseline leukotriene generation (Figure closed circles), Figure respectively. ALA (18:3n-3), present in echium supplementation with BO and EO resulted in decreased oil, tended to increase in all groups during the supplemen- FcεRI-mediated leukotriene generation. This inhibitory tation period (weeks 1–3) and was significantly higher effect was statistically significant in all groups during

Arm et al. Lipids in Health and Disease 2013, 12:141 Figure 2 Effects of dietary supplementation with borage andechium seed oils on concentrations of plasma n-3 fatty acids.
Data are expressed as a percentage of total plasma fatty acids andare shown for ALA (open bar), SDA (black bars), EPA (gray bars), DPA(cross-hatched bars), and DHA (white striped bars). Fatty acid profiles were monitored during the time course of the study beginning atbaseline (Pre), during supplementation (weeks 1–3) and during thewashout phase (w/o1-3). Data are mean ± SEM for each group.
Statistically significant rises in SDA, EPA and DPA were noted overtime (p < 0.0001) with significant differences amonggroups (p = 0.004).
treatment with BO and EO (p < 0.0001) with a significantdifference between study groups (p < 0.0001 for differ- ences among groups across time). The most stable and ro- w/o 1 w/o 2 w/o 3
bust inhibition of leukotriene generation was seen inGroup 2 (Figure , in which individuals ingested EO/BO combination that provided 1.7 g of GLA, 2.01 g ALA and 0.9 g SDA daily. In this group there was a time-dependentinhibition of cysteinyl leukotriene generation from baso- phils, stimulated with all concentrations of the antibody to εRI that reached 57% and 50% inhibition of the maximal response by weeks 2 and 3, respectively. A similar inhib- ition of FcεRI-dependent cysteinyl leukotriene generation was observed in Group 3 (Figure in which individuals ingested a BO/EO combination that provided 1.6 g of w/o 1 w/o 2 w/o 3
GLA, 1.15 g ALA and 0.5 g SDA daily (n = 8). In Group 3,inhibition was maximal (60%) at week one and cysteinyl leukotriene generation remained suppressed at weeks 2 and 3. Inhibition of FcεRI-dependent cysteinyl leukotrienegeneration from peripheral blood basophils was less ro- bust in Groups 1 and 4.
The inhibitory effect of dietary BO and EO on baso- philic leukotriene generation was transient. Figure shows the reversal of ex vivo cysteinyl leukotriene gener- ation from basophils (stimulated with a maximum con- w/o 1 w/o 2 w/o 3
centration of stimulating antibody) during the washoutperiod after individuals stopped taking capsules of bor- age and echium oil supplements. Data are expressed aspercentage of the maximum cysteinyl leukotriene gener- ation prior to starting supplements. In all groups, therewas a return to baseline values within 3 weeks.
A23187 is a calcium ionophore that stimulates leukotri- ene generation by eliciting calcium flux in a receptor- independent manner. In neutrophils, it provides a robuststimulus for leukotriene generation and allows assessment of the integrity of the whole leukotriene biosynthetic path-way including the terminal product, LTB4, and the stable non-enzymatic degradation products of its intermediates, w/o 1 w/o 2 w/o 3
5-HETE (from 5-hydroperoxyeicosatetraenoic acid) and all-trans-LTB4 (from LTA4). We therefore examined theeffects of dietary supplementation with BO and EO onA23187-stimulated leukotriene generation from neutro-phils. The shape and position of the dose response curvesfor release of LTB4 and 5-HETE were similar at baseline, Arm et al. Lipids in Health and Disease 2013, 12:141 Figure 3 Effects of dietary supplementation with borage and echium seed oils on concentrations of plasma n-6 fatty acids.
Data are expressed as a percentage of total plasma fatty acids and are shown for GLA (black bars), DGLA (white bars), and AA (graybars). Fatty acid profiles were monitored during the time course of the study beginning at baseline (Pre), during supplementation (weeks 1–3) and during the washout phase (w/o1-3). Data are mean± SEM for each group. Statistically significant rises in GLA and DGLA were noted over time (p < 0.0001) with no statistically significantdifferences among groups.
and changes in generation of LTB4 and 5-HETE during w/o 1 w/o 2 w/o 3
dietary supplementation with BO and EO were similar(data not shown). Therefore, data are presented as total Group 2
leukotriene generation; i.e. the sum of 5-HETE, LTB4, and all-trans-LTB4. A23187 elicited the dose-dependent re- lease of leukotrienes that was apparent at a concentration of 0.3 μM and reached a maximum response at a concen-tration of 3 to 10 μM A23187 (Figure . The quantities of all-trans LTB4 diastereoisomers that were generated were small and were not always readily measured by UV ab- sorbance; they are included in the data where possible.
Treatment with BO/EO combinations led to a significant w/o 1 w/o 2 w/o 3
inhibition of A23187-stimulated leukotriene generation from neutrophils (p < 0.0001) with a significant difference Group 3
in effect between groups (p = 0.02 for differences among groups across time). The group-dependent effects of diet- ary supplementation with BO and EO on A23187- stimulated leukotriene generation from neutrophils were similar to those observed for FcεRI-dependent cysteinyl leukotriene generation from basophils (Figure ). The greatest effect was seen in Group 2 (Figure ), in which maximal total leukotriene generation was inhibited 43%after three weeks of dietary supplementation with BO/EO combinations. The inhibitory effect of the dietary supple- w/o 1 w/o 2 w/o 3
mentation on neutrophil leukotriene generation declined Group 4
over time in Group 3 and was less robust in Group 4 (Figure ). As in the case of basophils, A23187-stimulated leukotriene generation from neutrophils returned to base- line during the 3-week wash-out period (data not shown).
Botanical seed oils from plants such as borage andechium have shown modest efficacy in a number of ani- mal and human inflammation models and disease. These botanicals contain 18C-PUFAs (ALA, SDA and GLA) that can be metabolized into 20–22 carbon PUFAs suchas EPA, DHA, DGLA and AA. All of these have beenshown to impact eicosanoid generation. However, a bet-ter understanding of the in vivo biochemistry of poten-tially bioactive PUFAs found in these botanical seed oilsand oil combinations and their capacity to block inflam-matory processes including eicosanoid production isneeded to enhance the effectiveness of botanical seed Arm et al. Lipids in Health and Disease 2013, 12:141 Figure 4 Dietary supplementation with borage and echium seed oils decreases FcεRI-dependent cysteinyl leukotriene generation by peripheral blood basophils. Cysteinyl leukotriene generation is shown prior to (, closed circles) and one week (Δ, open triangles), two weeks (□, open squares), and three weeks (○, open circles) after dietary supplementation with one of four borage and echium seed oil combinations (Groups 1–4, respectively) in response to buffer alone, control IgG1 (1.0 μg/ml, IgG), and 15A5, an activating antibody against FcεRI (0.01 to 1.0 μg/ml). Due totechnical errors, data are not available for one subject in each of groups 1, 3, and 4. In addition, basophils from one subject in eachof groups 3 and 4 failed to release leukotrienes upon stimulation, a well described phenomenon due to impaired signaling through Syk.
Data are expressed as a percentage of maximal cysteinyl leukotriene generation in each subject and are expressed as means ± SEM.
Statistically significant suppression of ex vivo leukotriene generation was noted (p < 0.0001) with a significant difference between groups(p < 0.0001).
oils. The current study utilized various BO/EO combina- tions to understand these processes.
Supplementation with BO/EO combinations increases plasma levels of n-3 and n-6, 18 carbon and 20–22 carbon PUFAs during the supplementation periods (Figure and Figure . Of note, circulating levels of three PUFAs,DGLA, EPA and DPA increased after supplementation. It is likely that DGLA increased as a result of GLA found in both BO and EO. As discussed above, GLA is readily elon- gated to DGLA in cells and tissues utilizing an enzyme encoded for by a gene known as elongase 5 (ELOVL5).
Once formed, DGLA is incorporated into inflammatory cells and tissues and competes with AA for the actionof cytosolic phospholipase A2 and cyclooxygenase to 3wks supplementation
Wk 1 washout
Wk 2 washout
Wk 3 washout
% of Pre-Diet Maxim
Figure 5 Cessation of dietary supplementation reversesinhibition of FcεRI-dependent cysteinyl leukotriene generationby peripheral blood basophils. After 3 weeks of dietarysupplementation (open bars), subjects stopped ingesting borageand echium seed oils. FcεRI-stimulated basophilic cysteinylleukotriene generation was measured at one (gray bar), two(cross-hatched bar) and three weeks (solid bar) of washout. Data areshown for maximal cysteinyl leukotriene generation in response to1.0 μg/ml 15A5. Data are expressed as a percentage of maximalcysteinyl leukotriene generation in each subject and are expressedas means ± SEM.
Arm et al. Lipids in Health and Disease 2013, 12:141 Figure 6 Dietary supplementation with borage and echium oilsdecreases A23187-stimulated total leukotriene generation by Group 1
peripheral blood neutrophil. Leukotriene generation is shownprior to (, closed circles) and one week (Δ, open triangles), two weeks (□, open squares), and three weeks (○, open circles) after dietary supplementation with one of four borage and echium seed oil combinations (Groups 1–4, respectively) in response to buffer alone and increasing concentrations of the calcium ionophore, A23187 (0.1 to 10 μM). Total leukotriene generation is the sum ofLTB4, 5-HETE, and, where measurable, all-trans-LTB4 isomers. Data are expressed as means ± SEM. Statistically significant suppression of ex vivo leukotriene generation was noted (p < 0.0001) with asignificant difference in effect between groups (p = 0.02).
form PGE1. Additionally DGLA is converted to a 15- Group 2
lipoxygenase product, 15-hydroxyeicosatrienoic acid (15 HeTrE) by human mononuclear leukocytes 15-HETrE has been demonstrated to be a potent blocker of LTB4 formation.
Additionally this botanical oil combination increased circulating levels of EPA and DPA This contribution waslike due to EO addition since it contains the precursor PUFAs, ALA and SDA. Providing 0.25 g/d to 1.75 g/d of otrienes
Buffer 0.1
SDA and 0.57 g/d to 4.02 g/d of ALA from EO led tosignificant and dose-dependent increases in circulating EPA and DPA; plasma EPA concentrations rose morethan 2-fold in the group receiving the highest concentra- tion of SDA (Figure This increase in EPA and DPA is Group 3
likely a result of SDA and not ALA as in vivo SDA con- version to EPA is 4–5 fold more efficient than ALA.
However, some epidemiological studies suggest that ALA-containing oils (from seed oils such as flax; Linumusitatissimum L.) ] may provide independent protec- tion from cardiovascular disease.
Numerous studies show the biological impact of EPA and DPA. EPA reduces AA metabolism through several Buffer 0.1
mechanisms including decreasing AA mobilization from membrane phospholipids, competition for cylooxygenaseand 5-lipoxygenease and reducing the expression of AA metabolizing enzymes and proinflammatory cytokines.
Group 4
Additionally, EPA can serve as a substrate for prostaglan- din formation generating "3-series" prostaglandin products including PGD3, PGE3, PGF3α, PGI3, and TxA3 and "5series" leukotriene products including LTB5 and LTC5 [. Reduced asthma symptoms with n-3 PUFA ingestionhave been shown to be related to 5-series leukotriene production []. With regard to inflammation, DPA is A21387 (µM)
beginning to receive attention. DPA is converted to 11-hydroxy-7,9,13,16,19-DPA and 14-hydroxy7,10,12,16,19-DPA, which inhibit aggregation of platelets and contain10-fold greater capacity to elicit endothelial cell migrationthan EPA, a biological process critical to wound healing[]. There were no changes in plasma levels of DHA,likely reflecting the poor bioconversion of EPA to DHA.
Arm et al. Lipids in Health and Disease 2013, 12:141 Previous studies have shown that GLA-containing oils combinations led to a significant inhibition of A23187- such as BO have the potential to increase circulating AA stimulated leukotriene generation (Figure that was which could enhance inflammation and platelet aggrega- almost as great as the inhibition of basophil cysteinyl leu- tion through increased thromboxane formation kotriene generation (Figure We recently reported that However, there were no changes in AA levels as a result dietary supplementation with BO and fish oil led to re- of BO/EO supplementation. It is possible that the ob- duced expression of phosphatidylinositol 3-kinase, a key served increase in EPA resulting from of the botanical signaling molecule, in circulating mononuclear cells combination is a feedback inhibitor of AA production It is therefore possible that the inhibition of cysteinyl leu- via the Δ5 desaturation step. EPA has been demon- kotriene generation that we observed in basophils was strated to inhibit the in vivo and in vitro desaturation of due, at least in part, to inhibition of signaling through DGLA to form AA In any event, the BO/EO FcεRI. However, the inhibition of leukotriene biosyn- combination led to an increase in three 20–22 carbon thesis in neutrophils stimulated through A23187, a re- PUFAs, DGLA, EPA and DPA that have been demon- ceptor independent stimulus, argues for a more direct strated to inhibit AA metabolism and attenuate inflam- effect of BO and EO on leukotriene biosynthesis.
mation without increasing circulating levels of AA.
The final objective of this paper was to determine whether these botanical oil combinations had the capacity Our data demonstrate that ingestion of certain combina- to inhibit leukotriene generation from two inflammatory tions of BO and EO increases circulating levels of both cells, basophils and neutrophils, isolated from subjects n-6 and n-3, 20–22 carbon PUFAs. Supplementation of with mild asthma who had supplemented their diet with human and animal diets with these fatty acids has been BO/EO. Prior studies of dietary supplementation with shown to inhibit metabolism of AA to pro-inflammatory GLA have demonstrated a reduction in ex vivo leukotriene lipid mediators and ameliorate inflammatory disease generation in whole blood or neutrophils stimulated with models including asthma, arthritis and coronary artery calcium ionophore A23187 or with zymosan ,].
disease These same combinations do not increase Basophils [], IgE ], and cysteinyl leukotrienes [] circulating AA levels, as has been observed with BO have been strongly implicated in the pathobiology of alone. Importantly, the observed biochemical changes asthma. We therefore assessed the effects of BO/EO com- occur concomitantly with a reduced capacity of inflam- binations on the generation of cysteinyl leukotrienes from matory cells from mild asthmatics supplemented with basophils stimulated through the high affinity IgE re- BO/EO combinations to produce leukotrienes that have ceptor, a physiologically relevant stimulus for asthma.
been illustrated to be important to promoting the asth- Significant inhibition of basophil cysteinyl leukotriene matic response. Consequently, an important next step generation was noted within one week of dietary supple- would be to determine whether such BO/EO combina- mentation (Figure . Interestingly, the time dependence tions have the capacity to improve clinical symptoms of of this inhibition varied between groups. The least robust inhibition was observed in Group 4, in which subjects re-ceived the lowest dose of SDA. Although there is consid- erable variation in the extent of inhibition of ex vivo AA: Arachidonic acid; ALA: α linolenic acid; BLT1: B leukotriene receptor 1; leukotriene generation among individuals and the groups BO: Borage oil; CysLT1R: Cysteinyl leukotriene receptor 1; CysLT2R: Cysteinylleukotriene receptor 2; DGLA: Dihommo-gammalinolenic acid; were relatively small, the between group variation was sta- DHA: Docosahexaenoic acid; DPA: Docosapentaenoic acid; EO: Echium oil; tistically significant. Furthermore, a comparable variation EPA: Eicosapentaenoic acid; GLA: γ linolenic acid; GPCR: G protein coupled in supplementation-induced inhibition of leukotriene gen- receptor; IND: Investigational new drug; LTB4: Leukotriene B4;LTC4: Leukotriene C4; LTD4: Leukotriene D4; LTE4: Leukotriene E4; eration was observed in response to A23187- stimulation PBMC: Peripheral blood mononuclear cells; SDA: Stearidonic acid.
of neutrophils (Figure . The data therefore suggest thatproviding SDA in the diet contributed to the extent of in- Competing interests hibition of leukotriene generation, consistent with data FHC is an unpaid consultant for Gene Smart Health and receives nocompensation or equity in this role. This information has been disclosed to showing that dietary supplementation with EPA leads to WFUHS and outside sponsors, as appropriate, and is institutionally managed.
inhibition of ex vivo leukotriene generation .
All other authors declare no competing interest.
In a receptor-independent manner, A23187 robustly stimulates human neutrophils to elicit maximal gener- Authors' contributionsJPA and FHC designed the study and protocols, oversaw the conduct of the ation of LTB4, the product of leukotriene biosynthesis study and wrote the manuscript; EI participated in the conduct of the study; in neutrophils. Utilization of this stimulus allows an as- LW and HC performed ex vivo experiments and sample analyses; PI and KLW sessment by RP-HPLC of the non-enzymatic degradation analyzed fatty acids and biomarkers; MZ and VP were study coordinators; UGperformed statistical analyses; JAB and SS helped with data analysis and products of the proximal intermediates of leukotriene interpretation and manuscript preparation. All authors read and approved biosynthesis. Dietary supplementation with BO/EO the final manuscript.
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