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Marine lipid fraction pcso-524™ (lyprinol®/omega xl®) of the new zealand green lipped mussel attenuates hyperpnea-induced bronchoconstriction in asthma

Available online at Marine lipid fraction PCSO-524 (lyprinol/omega XL) of the New Zealand green lippedmussel attenuates hyperpnea-inducedbronchoconstriction in asthma Timothy D. MickleboroughCherissa L. Vaughn, Ren-Jay Shei,Eliza M. Davis, Daniel P. Wilhite School of Public Health-Bloomington, Department of Kinesiology, Human Performance and ExerciseBiochemistry Laboratory, 1025 E. 7th St. SPH 112, Bloomington, IN 47404, USA Received 12 February 2013; accepted 10 April 2013 Omega-3 fatty acids; Purpose: Evaluate the effect of the marine lipid fraction of the New Zealand green-lipped mussel (Perna canaliculus) PCSO-524 (Lyprinol/Omega XL), rich in omega-3 fatty acids, on airway inflammation and the bronchoconstrictor response to eucapnic voluntary hyperpnea (EVH) in asthmatics.
Methods: Twenty asthmatic subjects, with documented HIB, participated in a placebocontrolled double-blind randomized crossover trial. Subjects entered the study on their usualdiet and were then placed on 3 weeks of PCSO-524 or placebo supplementation, followed bya 2 week washout period, before crossing over to the alternative diet. Pre- and post-eucapnicvoluntary hyperpnea (EVH) pulmonary function, fraction of exhaled nitric oxide (FENO),asthma symptom scores, medication use, exhaled breath condensate (EBC) pH, cysteinyl leu-kotrienes (cyst-LT), 8-isoprostane and urinary 9a, 11b-prostaglandin (PG)F2 and Clara (CC16)protein concentrations were assessed at the beginning of the trial and at the end of each treat-ment period.
Results: The PCSO-524 diet significantly reduced (p < 0.05) the maximum fall in post-EVHFEV1 (8.4  3.2%) compared to usual (19.3  5.4%) and placebo diet (22.5  13.7%).
Pre- and post- EVH EBC cyst-LT and 8-isoprostane, and urinary 9a, 11b-PGF2 and CC16 concen-trations were significantly reduced (p < 0.05) on the PCSO-524 diet compared to the usualand placebo diet. EBC pH and asthma symptom scores were significantly improved(p < 0.05) and rescue medication use significantly reduced (p < 0.05) on the PCSO-524 dietcompared to the usual and placebo diet.
* Corresponding author. Tel.: þ1 812 855 0753; fax: þ1 812 855 3193.
E-mail address: (T.D. Mickleborough).
0954-6111/$ - see front matter ª 2013 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Mickleborough TD, et al., Marine lipid fraction PCSO-524 (lyprinol/omega XL) of the New Zealandgreen lipped mussel attenuates hyperpnea-induced bronchoconstriction in asthma, Respiratory Medicine (2013), T.D. Mickleborough et al.
Conclusion: PCSO-524 (Lyprinol/Omega XL) may have beneficial effects in HIB and asthmaby serving as a pro-resolving agonist and/or inflammatory antagonist.
ª 2013 Elsevier Ltd. All rights reserved.
with similar potency to low dose n-3 PUFA supplementa-tion.These findings support the potential for PCSO-524 Asthma is a multifaceted condition in which multiple to attenuate airway inflammation and bronchoconstriction environmental and genetic influences can lead to several clinical phenotypes such as exercise-induced bronchocon- Daily supplementation with PCSO-524 in human asth- striction (EIB), which is a transient deterioration in lung matics has been shown to decrease daytime wheeze and function following exercise,that can occur in patients with exhaled breath hydrogen peroxide concentration (a marker and elite athletes.The mechanisms responsible of airway inflammation),and to suppress the develop- for EIB likely involve multiple mechanistic pathways, how- ever it is generally accepted that exercise or dry air hy- responsiveness in a mouse model of ovalbumin (OVA)- perpnea play an important role as an initiating stimulus induced allergic airway disease.
through airway surface effects of water loss, which include Therefore, the primary aim of this study was to evaluate mucosal cooling, dehydration and epithelial the effects of PCSO-524 supplementation on airway This transient dehydration causes an increase in airway inflammation and the bronchoconstrictor response to dry surface liquid osmolality which activates histamine, neu- air hyperpnea in individuals with asthma. We hypothesized ropeptides, and the release of leukotrienes and prosta- PCSO-524 supplementation would significantly glandins (arachidonic acid metabolites), from resident attenuate airway inflammation and hyperpnea-induced bronchoconstriction (HIB) in individuals with asthma.
contraction and subsequent airway Although the treatment of EIB almost exclusively involves pharmacotherapy, there is mounting evidence that nutri- tional supplementation has potential to modify this condi-While the clinical data on the effect of fish oil supplementation in asthma has been supple-menting the diet with fish oil rich in omega-3 (n-3) poly- Twenty subjects (12 males, 8 females, aged 22.6  2.1 yr, unsaturated fatty acids (PUFA), such as eicosapentaenoic height 168.8 þ 11.2 cm) with both physician-diagnosed acid (EPA) and docosahexaenoic acid (DHA), in asthmatic asthma and documented HIB were recruited from a popu- eand elite athletes with has yielded lation of university students and the local community promising results. Two mechanisms of action underpinning All subjects had clinically treated mild to moderate the novel anti-inflammatory bioactions of fish oil include the persistent asthma, with a resting forced expiratory volume ability of EPA to compete with arachidonic acid as a substrate in 1-sec (FEV1) of >65% predicted (and EIB as for cyclooxygenase (COX)-2 and 5-lipoxygenase (5-LO) en- demonstrated by a greater than 10% drop in FEV1 following zymes and be converted to less inflammatory leukotrienes a eucapnic voluntary hyperventilation (EVH) A and prostanoids,and to generate the potent anti- group of non-asthmatic (control) subjects was not included inflammatory E-series resolvins.Less well-characterized in the present study, as it has been shown that n-3 PUFA mechanisms include the capability of DHA to alter gene supplementation does not alter pulmonary function or in- transcription and translation via direct or indirect actions on flammatory mediator generation in this population.
intracellular signaling and to produce the D- All subjects had a history of shortness of breath, chest series resolvins and protectins (neuroprotectin D1).
tightness, and intermittent wheezing following exercise, While fish oil has been shown to attenuate airway which was relieved by bronchodilator therapy. No subjects inflammation and the bronchoconstrictor response to ex- who volunteered for the study were currently taking any ercise and dry gas hyperpnea,it is possible that maintenance medications (e.g., corticosteroids and leuko- different forms of marine oils in the diet may have varying triene modifiers) for asthma. Short acting b2-agonists were effects of on these responses, since these oils contain a discontinued 12 h prior to testing. Caffeine/alcohol and variety of lipid mediators as well as different amount of n-3 physical exercise was not permitted 8 h and 12 h respec- PUFAs.PCSO-524 (Lyprinol/Omega XL) is a patented tively prior to the EVH challenge. Subjects were also extract of stabilized lipids from the New Zealand green- excluded if they had a history of taking fish oil supplements lipped mussel (NZGLM), Perna canaliculus, combined with and regularly consumed more than one fish meal per week.
olive oil and vitamin E.PCSO-524 is a multifarious Subjects were asked not to eat more than one fish meal per mixture of sterol esters, sterols, polar lipids, triglycerides week during the course of the study. Subjects were and free fatty acids (including EPA and and has excluded if they were pregnant, had a history of hyperlip- been shown to reduce pro-inflammatory leukotriene (LT)B4 idemia, hypertension, diabetes, bleeding disorders, or in human monocytes,and to decrease levels of throm- delayed clotting time. The study was approved by the boxane B2, prostaglandin (PG) E2, and interleukin (IL) 1b Indiana University Institutional Review Board for Human Please cite this article in press as: Mickleborough TD, et al., Marine lipid fraction PCSO-524 (lyprinol/omega XL) of the New Zealandgreen lipped mussel attenuates hyperpnea-induced bronchoconstriction in asthma, Respiratory Medicine (2013),

Omega-3 fatty acids and asthma CONSORT flow of participants through the study diagram.
Pre-hyperpnea (baseline) pulmonary function.
Definition of abbreviations: FVC, forced vital capacity; FEV1, forced expiratory volume in 1-s; FEF25e75%, forced expiratory flow at25e75% of FVC; PEF: peak expiratory flow. Values are mean  SD. There was no significant difference (p > 0.05) for any variablesbetween diets.
Please cite this article in press as: Mickleborough TD, et al., Marine lipid fraction PCSO-524 (lyprinol/omega XL) of the New Zealandgreen lipped mussel attenuates hyperpnea-induced bronchoconstriction in asthma, Respiratory Medicine (2013), T.D. Mickleborough et al.
Subjects, and written informed consent for all subjects was Eucapnic voluntary hyperventilation obtained prior to participation in the study. The study wasregistered as a phase 1 clinical trial with clinical The EVH protocol required subjects to breathe compressed (study # NCT01504646).
dry air (<3 mg H2O.L1 air and 21%O2, 5%CO2, balance N2)at a predetermined rate of 85% of maximal voluntary ventilation (estimated from 30  the volume of restingFEV1) for 6 min. Gas flowed from a cylinder to a reservoir The study was conducted as a randomized, double-blind, bag through high-pressure tubing. From the reservoir bag placebo-controlled crossover trial over 8 consecutive gas was directed to the subject through a tube connected weeks, with each subject serving as their own control.
to a two way breathing valve and mouthpiece. Expired Subjects were enrolled while on their usual diet. The order gases passed through a flow sensor and ventilation was of supplementation was randomly assigned with the use of measured and recorded as verification of respiration in- a computerized random number generator tensity (Vmax 22 Metabolic Measurement Cart, Sensor- ). The randomization sequence Medics, Yorba Linda, CA).
was created using a fixed random block size of two tocorrespond to the two treatments (i.e. PCSO-524 and Pulmonary function tests placebo). Sealed capsule bottles labeled with one of twomaterial numbers were provided. Data collection and initialdata analysis was completed before the principle investi- Pulmonary function tests were conducted on all subjects gator was informed which material number corresponded to each treatment. The active and placebo capsules were spirometer (Vmax 22, SensorMedics, Yorba Linda, CA) ac- identical in appearance and taste so that subjects were not cording to according to American Thoracic Society (ATS) aware of which treatment they received.
recommendations.The maximum percentage fall in FEV1 All subjects (n Z 20) underwent a 3 eweek run period from the baseline (pre-EVH) value was calculated using the (usual diet; phase 1) preceding the start of the trial in following equation: (Pre-EVH FEV1 e lowest post-EVH which asthma symptoms, bronchodilator use and peak flow FEV1)/(Pre-EVH FEV1). In addition, the bronchoconstrictor measurements were recorded, after which they were response to EVH was assessed as the area under the curve randomly assigned to receive either 8 capsules per day of of the percentage fall in post-EVH FEV1 plotted against time PCSO-524 (Lypriniol/Omega-XL; Pharmalink Interna- for 20 min (AUC0e20), using trapezoidal integration.
tional Ltd, Hong Kong) (n Z 10) containing approximately72 mg EPA and 48 mg DHA (1 capsule contains 50 mg n-3 Fraction of exhaled nitric oxide PUFAs and 100 mg olive oil or identical placebo (n Z 10,placebo diet) capsules containing 150 mg of olive oil for 3 Fraction of exhaled nitric oxide (FENO) was measured with weeks (phase 2). Thereafter, they followed a 2-week an online measurement of resting values using a restricted washout period (usual diet; phase 3) and then switched to exhaled breath protocol (NOA 280i Nitric Oxide Analyzer, the alternative diet for the remaining 3 weeks (phase 4). All Accurate NO Breath Kit, Thermal Mass Flowmeter, NO subjects were asked to record bronchodilator use, symptom scores and peak flow measurements during each dietary Boulder, CO). Measurements were conducted as outlined by treatment period.
American Thoracic Society guidelines.Three exhalations A eucapnic voluntary hyperventilation (EVH) challenge were performed with nose clips at each test with at least (surrogate for an exercise challenge test) was performed at 30 s between The procedure entailed the beginning of the study and at the end of each treatment maximal inhalation to total lung capacity and immediate period. Pulmonary function measurements were conducted exhalation against expiratory resistance for at least 6 s to pre-EVH and post-EVH at 5, 10, 15, and 20 min. Exhaled obtain a NO plateau lasting at least 3 s. Subjects were fraction of nitric oxide (FENO), a non-invasive measure of instructed to maintain a flow rate of 50  10 mL/s as airway inflammation, was measured pre-EVH and at 30 min monitored by a visual computer display.
post-EVH. Exhaled breath condensate (EBC) was collectedpre-EVH and post-EVH from 0 to 10 min and analyzed for thepresence of the cysteinyl (Cyst)-leukotrienes (LTs) (marker Quantification of exhaled breath condensate of airway inflammation), 8-isoprostane (marker of oxida- tive and airway pH. Urine samples were collectedpre-EVH and 60 min post-EVH for 9a, 11b-Prostaglandin F2 EBC samples were collected with a specially designed (sensitive marker of mast cell activation and airway inflam- condensing chamber (ECoScreen, Jaeger, Hoechberg, Ger- mation), and pneumoprotein Clara cell (CC16; marker of many) using ATS/ERS recommendations.The EBC protocol airway epithelial stress) analysis. Food frequency ques- required subjects to breathe normally, wearing nose clips, tionnaires were administered at the beginning of each through a mouthpiece connected to a non-rebreathing testing session. Pre- and post-EVH pulmonary function and valve, whereby exhaled breath entered a condenser sys- FENO measurements were conducted at the end of the 2- tem. A temperature of 20 C inside the condensing week washout period in order to verify that lung function chamber, which was maintained throughout the collection and exhaled nitric oxide values had returned to baseline time, produced immediate sample freezing. Exhaled breath levels. Subjects were asked to record daily peak flow and was collected for 10 min prior to and intervals (0e10 min) asthma symptom scores throughout the course of the study.
following the EVH challenge.
Please cite this article in press as: Mickleborough TD, et al., Marine lipid fraction PCSO-524 (lyprinol/omega XL) of the New Zealandgreen lipped mussel attenuates hyperpnea-induced bronchoconstriction in asthma, Respiratory Medicine (2013), Omega-3 fatty acids and asthma The pH of the non-deaerated EBC was measured imme- acids, saturated fatty acids). Adherence to the treatment diately following collection (Orion 2 star pH meter, Thermo regimen was monitored by asking the subjects to document Scientific, Beverly, MA). It has been shown that pH mea- the dose of capsules consumed daily and to return any un- surements in EBC collected by ECoScreen are repeatable used capsules.For the purpose of the study a compliance of and reproducible.
90% was considered acceptable.
The remainder of the condensate was stored at 80 C for later analysis of Cyst-LTs and 8-isoprostane using Symptoms, rescue b-agonist use and peak flow enzyme immunoassay techniques (Cayman Chemicals, Ann Arbor, MI) as previously Cross-reactivity of theCyst-LT antibody against an array of related compounds is: Symptoms such as wheeze, shortness of breath, chest LTC4 (100%), LTD4 (100%), LTE4 (67%), LTD5 (61%), LTC5 tightness, and cough were recorded by each subject once (54%), LTE5 (41%), N-acetyl-LTE4 (10.5%) and below 0.01% daily with the use of a logbook. The symptom severity for other primary eicosanoid metabolites. The intra- and rating scale was defined as follows: 0: absent, no symp- inter-assay coefficient of variation (CV) for the Cyst-LT toms; 1: mild, symptom was minimally troublesome (not enzyme immunoassay kit is reported to be <10% respec- sufficient to interfere with normal daily activity or sleep); tively. Cross-reactivity of the 8-isoprostane antibody 2: moderate, symptom was sufficiently troublesome to against an array of related compounds is: 8-isoprostane interfere with normal daily activity or sleep; 3: severe, (100%), 8-iso Prostaglandin F2a ethanolamide (100%), and symptom was so severe as to prevent normal activity and/ 8-iso Prostaglandin F3a (20.6%).
or sleep. All subjects were instructed to record each use oftheir rescue medication throughout the study duration, Urinary 9a, 11b-prostaglandin (PG) F2 and clara cell entering each medication use and the number of puffs used (CC16) quantification per occasion. Subjects were asked to perform 3 peak flowmaneuvers at home in the morning and evening 2 weeks Urine collection containers were provided to each subject prior to the start of study, and throughout the course of the during each visit to the laboratory. The urine was subse- study. The subjects were provided with a peak flowmeter quently pipetted into microfuge tubes and stored at 80 C (Piko-1, Ferraris, Louisville, CO) and a log to record the best until analysis. Urine was assayed for 9a, 11b-PGF2 (Cayman of 3 trials.
Chemicals, Ann Arbor, MI) and Clara Cell protein (CC16)(BioVendor LLC, Karasek, Czech Republic) using enzyme immunoassay techniques as discussed previouslyThe9a, 11b-PGF2 antibody cross-reacts with 2,3 dinor-11b- The primary analysis was on an intention-to-treat basis and PGF2a (10%), 11b-13,14-dihydro-15-keto-PGF2a (0.5%) and involved all subjects who were randomly assigned. Data below 0.01% for all other primary eicosanoid metabolites.
were analyzed using the SPSS version 20.0 statistical soft- The antibodies used in the CC16 ELISA are specific for ware (SPSS Inc., Chicago, USA). Normality of data was human Clara cell protein with no detectable cross reac- assessed using a KolmogoroveSmirnov test and Levene's tivity's to the cytokines that may be present in human test was used to check for homogeneity of variance be- serum. Inter- and intra-assay CV for the 9a, 11b-PGF2 and tween groups. A two-way repeated measures analysis of CC16 assay is <15%, and 2.2% and 3.7% respectively. The variance (ANOVA) was used to analyze the data, with both concentration of 9a, 11b-PGF2 and CC16 was adjusted for treatment and time as "within-subject" effects, whereas a creatinine concentration (Cayman Chemicals, Ann Arbor, two-way analysis of variance was used to analyze "be- MI). The intra- and inter assay CV for creatinine is reported tween-subject" effects. Mauchly's test was conducted to to be 2.7% and 3.0% respectively.
determine if sphericity was violated. If sphericity wasviolated, the repeated measures ANOVA were corrected Nutrient intake and compliance using a Greenhouse-Geisser adjustment factor. Where asignificant F ratio was found (p < 0.05), a Fisher protected Nutrient intake was monitored to ensure that dietary factors least-square difference post-hoc test was used to detect that could potentially affect asthma or EIB did not change differences in group means (p < 0.05). The Wilcoxon signed through the course of the study. Nutrient data was collected rank test was used to compare symptom scores and bron- using the GSEL food frequency questionnaire developed by chodilator use during the trial. Data were analyzed for the the Nutrition Assessment Shared Resource (NASR) of Fred presence of carryover effects between treatments using a Hutchinson Cancer Research Center. This questionnaire has 2  2 ANOVA. Statistical significance was set at p < 0.05.
been shown to be valid and reliable in the collection of di- Data are expressed as mean  SD, and their 95% confidence etary Subjects completed the GSEL version of the interval (CI).
questionnaire at the first testing session and at the end ofeach supplementation period. Analysis of GSEL for nutrient intake was conducted by the Fred Hutchinson CancerResearch Center. Nutrients of interest obtained from theGSEL analysis included macronutrient composition, antioxi- dants (a-tocopherol, b-carotene, lycopene, Vitamin C),certain minerals (magnesium, sodium, zinc), and types of Bronchodilator use (average number of doses/puffs per dietary fatty acids (omega-3, total polyunsaturated fatty day) was significantly reduced during the last 2 weeks of Please cite this article in press as: Mickleborough TD, et al., Marine lipid fraction PCSO-524 (lyprinol/omega XL) of the New Zealandgreen lipped mussel attenuates hyperpnea-induced bronchoconstriction in asthma, Respiratory Medicine (2013),

T.D. Mickleborough et al.
the PCSO-524 diet (1.6  0.7 puffs) compared to thenormal diet [9.8  2.5 puffs; p < 0.001; D (mean differ-ence), 8.2  2.3 puffs; 95% CI, 6.3 to 8.8 puffs] and placebodiet (8.5  2.5 puffs; p < 0.001; D, 6.9  2.8 puffs; 95% CI,5.4 to 8.4 puffs). There was no significant difference(p Z 0.456) in bronchodilator use between the normal andplacebo diet. A significant improvement in mean asthmasymptoms scores during the 3 week treatment period wasobserved on the PCSO-524 diet (0.8  0.5) compared tothe normal diet (2.6  0.5; p < 0.001; D, 1.8  0.9; 95% CI,1.3e2.3) and placebo diet (2.8  0.4; p < 0.001; D,2.0  0.6; 95% CI, 1.6e2.3). No significant difference(p Z 0.423) in mean asthma symptom scores was observedbetween the normal and placebo diet. The combined meanmorning and evening peak flow was significantly increasedduring the 3 week treatment period on the PCSO-524 diet(386.3  22.8 L/min) compared to the normal diet(370.4  23.6 L/min; p Z 0.001; D, 15.9  16.2 L/min; 95%CI, 7.3 to 24.5 L/min) and placebo diet (364.5 þ 17.2 L/ The percentage change in FEV1 from pre- to post- min; p < 0.000; D, 21.8 þ 16.1 L/min; 95% CI, 13.2 to EVH across the three treatments. Reductions in post-EVH in excess of 10% represent abnormal pulmonary function. Letters (p Z 0.221) between the normal and placebo diet for the a and b refer to comparisons by treatment within respective combined mean morning and evening peak flow. A 2  2 time period. Different letters designate a significant difference ANOVA, used to test for the presence of carryover effects (p < 0.05).
between diets, indicated that none were present for allmeasured variables (p < 0.05); this was further supportedby post-EVH pulmonary function and F 33.6  33.7 ppb; p Z 0.046; D, 14.7  23.8 ppb; 95% CI, ENO values measured at the end of the 2-week washout period returning to 0.04 to 30.4; post-EVH: 27.8  28.0 ppb; p Z 0.035; D, baseline levels. In addition, no statistical difference 16.3  22.7 ppb; 95% CI, 0.9e28.4 ppb) and placebo diet (p > 0.05) was observed between sex for all dependent (pre-EVH: 25.2  19.1 ppb; p Z 0.048; D, 13.9  19.1 ppb; 95% CI, 0.2 to 27.6; post-EVH: 22.9  17.4 ppb; p Z 0.049; D, 11.4  17.3 ppb; 95% CI, 0.1e26.7 ppb) ( Pulmonary function Exhaled breath condensate and urinary No significant difference was observed (p > 0.05) was inflammatory markers observed in baseline (pre-EVH) pulmonary function be-tween groups (PCSO-524, placebo and usual diet) The EBC pre-EVH pH was significantly higher on the PCSO- The percentage change in the pre-EVH to post-EVH 524 diet (6.81  0.31) compared to the usual diet FEV1, as a consequence of diet, is shown in . Themaximum percentage drop in post-EVH FEV1 on the PCSO-524 diet (8.4  3.2%), which is indicative of an attenu-ated HIB response, was significantly less than the usual diet(19.3 þ 5.4%; p < 0.001; D, 10.9  5.6%; 95% CI, 13.9to 7.9%) and placebo diet (22.5  13.7; p < 0.001; D, 14.1  13.1%; 95% CI, 21.1 to 7.1%). Similar significant(p < 0.05) changes a result of diet were observed for thepresent drop in post-EVH FVC, FEF25e75% and PEF. Thebronchoconstrictor response to EVH as determined by theAUC0-20 for FEV1 was significantly less on the PCSO-524diet (112.3 þ 28.5) compared to the usual diet(296.8  33.4; p < 0.001; D, 184.5  26.4; 95% CI, 124.7 to 234.3) and placebo diet (338.6  38.6;p < 0.001, D, 226.3  31.2; 95% CI, 154.8 to 314.2). Asimilar pattern was observed for the AUC0e20 for post-EVHFVC, FEF25e75% and PEF.
Fraction of exhaled nitric oxide Mean fraction of exhaled nitric oxide (FENO) con- Pre- and post-EVH FENO levels were significantly reduced on (p < 0.05) from pre-EVH value within diet. * designates a sta- the PCSO-524 diet (pre-EVH: 15.3 þ 10.7 ppb; post-EVH tistical difference (p < 0.05) compared to respective time (11.5 þ 8.2 ppb) compared to the usual diet (pre-EVH: point between diet.
Please cite this article in press as: Mickleborough TD, et al., Marine lipid fraction PCSO-524 (lyprinol/omega XL) of the New Zealandgreen lipped mussel attenuates hyperpnea-induced bronchoconstriction in asthma, Respiratory Medicine (2013),

Omega-3 fatty acids and asthma (6.57 þ 0.29; p Z 0.016; D, 0.24 þ 0.35; 95% CI, 0.05e0.42) to the usual (pre-EVH: 41.2  11.2 pg/ml; p < 0.001; D, and placebo diet (6.60  0.25; p Z 0.042; D, 0.21  0.38; 10.7  4.8 pg/ml; 95% CI, 7.5e13.6 pg/ml; post-EVH: 95% CI, 0.00845e0.41) (Panel A). In addition, EBC pH 68.9  12.1 pg/ml; p < 0.001; D, 27.0  12.8 pg/ml; 95% was significantly increased on the PCSO-524 diet at 5 min CI, 17.4e34.7 pg/ml) and placebo (pre-EVH: 42.1  2 pg/ (6.99  0.47) and 10 min (7.11  0.51) post-EVH compared ml; p < 0.001; D, 11.6 þ 4.5 pg/ml; 95% CI, 7.8 to 13.8; to 5 min (6.66  0.29; p Z 0.037; D, 0.33 þ 0.59; 95% CI, post-EVH: 65.6  15.3 pg/ml; p Z 0.002; D, 23.7  14.9 pg/ 0.184e0.534) and 10 min (6.57  0.41; p Z 0.003; ml; 95% CI, 11.1e33.9 pg/ml) diet (Panel B).
0.54  0.55; 95% CI, 0.250e0.945) post-EVH on the usual Pre- and post-EVH EBC 8-isoprostane concentration was diet and compared to 5 min (6.56  0.36; p Z 0.046; D, significantly attenuated on the PCSO-524 diet (pre-EVH: 0.43  0.62; 95% CI, 0.214e0.844) and 10 min (6.71  0.31; 37.2  14.9 pg/ml; post-EVH: 53.8  13.3 pg/ml) compared p Z 0.015; D, 0.40  0.56; 95% CI, 0.134e0.938) post-EVH to the usual (pre-EVH: 46.4 þ 15.1 pg/ml; p Z 0.024; D, on the placebo diet.
9.2  13.4 pg/ml; 95% CI, 1.6e3.9 pg/ml; post-EVH: The mean pre- and post-EBC Cyst-LT concentration was 61.1  15.4 pg/ml; p Z 0.043; D, 7.3  13.2 pg/ml; 95% significantly reduced on the PCSO-524 diet (pre-EVH: 30.5  10.9 pg/ml; post-EVH: 41.9 þ 10.4 pg/ml) compared 49.3  15.5 pg/ml; p Z 0.001; D, 12.1  10.4 pg/ml; 95% Panel A. Mean exhaled breathe condensate (EBC) pH. # designates a statistical difference (p < 0.05) from pre-EVH value within diet. * designates a statistical difference (p < 0.05) compared to respective time point between diet. Panel B. Mean exhaledbreathe condensate (EBC) cysteinyl-leukotriene concentration (pg mg1). *, # designates a statistical difference (p < 0.05)compared to respective time point between diet. g designates a significant difference (p < 0.05) from pre-EVH value within diet.
Panel C. Mean exhaled breathe condensate (EBC) 8-isoprostane concentration (pg mg1). *, # designates a statistical difference(p < 0.05) compared to respective time point between diet. g designates a significant difference (p < 0.05) from pre-EVH valuewithin diet.
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T.D. Mickleborough et al.
CI, 7.2e21.2 pg/ml; post-EVH: 66.0 þ 13.1 pg/ml; period reflected that capsules were consumed on a regular p Z 0.040; D, 12.2  12.9 pg/ml; 95% CI, 0.63e20.5 pg/ml) basis. Compliance as estimated from return-tablet count diet , Panel C).
was high (median, 99%). Though the usual diet was ex- The mean urinary 9a, 11b PGF2 levels for pre- and post- pected to vary between and among subjects, mean daily EVH was significantly mitigated on the PCSO-524 diet nutrient intake of the subject's diets did not differ signifi-  8.1 ng/mg creatinine; post-EVH: cantly (p < 0.05) between the treatment regimens.
31.0  15.4 ng/mg creatinine) compared to the usual(pre-EVH: 23.6  11.9 ng/mg creatinine; p Z 0.036; D,4.8 þ 8.3 ng/mg creatinine; 95% CI, 0.36e9.1 ng/mg creatinine; post-EVH: 36.3  13.6 ng/mg creatinine;p Z 0.041; D, 5.3  11.7 ng/mg creatinine; 95% CI, 0.2e11.5) and placebo (pre-EVH: 26.2  13.8 ng/mg controlled study has shown that a diet supplemented with creatinine; p Z 0.008; D, 7.4  9.8 ng/mg creatinine; 95% PCSO-524 (Lyprinol/OmegaXL) a patented extract of CI, 2.2e12.5 ng/mg creatinine; post-EVH: 41.2  20.1 ng/ stabilized lipids from the NZGLM, P. canaliculus, attenuates mg creatinine; p Z 0.003; D, 10.2  11.6 ng/mg creatinine; airway inflammation, and the bronchoconstrictor response 95% CI, 3.9e16.3 ng/mg creatinine) diet , Panel A).
to dry gas hyperpnea, and can reduce bronchodilator use Mean urinary CC16 concentration pre- and post-EVH and asthma symptom scores in asthmatic subjects. The decreased significantly on the PCSO-524 diet (pre-EVH: PCSO-524 diet significantly reduced the severity of HIB as measured by AUC0e20, and significantly reduced the 0.69  0.49 ng/mmol creatinine) compared to the usual maximal fall in post-EVH FEV1 by approximately 57%. The (pre-EVH: 0.11  0.11 ng/mmol creatinine; p Z 0.048; D, degree of protection provided by PCSO-524 on HIB in the present study is similar in magnitude to previous reports showing that 3 weeks of fish oil, rich in n-3 PUFA, reduced  0.45 ng/mmol creatinine; p Z 0.017; D, the maximum fall in FEV1 post-exercise by almost 80%and 0.35  0.51 ng/mmol creatinine; 95% CI, 0.07e0.6) and in elite athletes and asthmatic subjects with EIB placebo (pre-EVH: 0.14  0.18 ng/mmol creatinine; respectively, and reduced the maximum fall in post-EVH p Z 0.048; D, 0.086  0.16 ng/mmol creatinine; 95% CI, FEV1 by approximately 49% in asthmatic subjects with HIB.
0.0003 to 0.172; post-EVH: 0.98 þ 0.44 ng/mmol creatinine; In the present study the PCSO-524 diet significantly p Z 0.043; D, 0.29 þ 0.51 ng/mmol creatinine; 95% CI, reduced the pre-EVH FENO compared to the placebo and 0.011e0.56) diet , Panel B).
usual diet, suggestive of amelioration in baseline airway There was no significant difference (p > 0.05) between inflammation; this finding supports previous observations the usual and placebo diets for pre-and post-EVH EBC Cyst- that n-3 PUFA supplementation can moderate baseline LT and 8-isoprostane, and urinary 9a, 11b PGF airway inflammation in In addition, the present study demonstrated that the PCSO-524 diet canreduce post-EVH FENO values compared to the usual andplacebo diets. It has been shown that FENO is an indirect Nutrient intake and compliance marker of asthmatic airway inflammation,and that arelationship exists between FENO levels and Subject adherence to the treatment regimens were assured It has been shown that airway pH is a significant deter- by finding that pill counts at the end of each treatment mining factor of expired FENO and airway inflammation, Panel A. Mean urinary 9a, 11b-prostaglandin F2 concentration (ng mg mmol creatinine1). *, # designates a statistical difference (p < 0.05) compared to respective time point between diet. g designates a significant difference (p < 0.05) from pre-EVH value within diet. Panel B. Mean urinary CC16 concentration (ng mmol creatinine1). *, # designates a statistical difference(p < 0.05) compared to respective time point between diet. g designates a significant difference (p < 0.05) from pre-EVH valuewithin diet.
Please cite this article in press as: Mickleborough TD, et al., Marine lipid fraction PCSO-524 (lyprinol/omega XL) of the New Zealandgreen lipped mussel attenuates hyperpnea-induced bronchoconstriction in asthma, Respiratory Medicine (2013), Omega-3 fatty acids and asthma and thus there may be a causal relationship between airway during exercise in vivo in elite swimmers, and following an acidification and airflow limitation in Airway pH isocapnic hyperpnea challenge in trained and untrained appears to be lower in asthmatics and correlates positively individuals, with and without EIB.
with sputum eosinophilia, total nitrate/nitrite, and oxida- In the present study the PCSO-524 diet significantly tive stress.While airway acidity both accelerates human blunted the increase of urinary 9a, 11b-PGF2 prior to and eosinophil necrosis and can cause the conversion of following the dry gas hyperpnea challenge, which confirms endogenous nitrate ðNOÞ to nitric oxide (NO), the acidic previous findings of attenuated urinary 9a, 11b-PGF airway breath condensate pH in asthma tends to normalize following 3 weeks n-3 PUFA supplementation in elite ath- with anti-inflammatory therapy,which supports the letes with EIBand asthmatics with 9a, 11b-PGF2, the findings of the present study, and our previous observa- urinary metabolite of PGD2, is a sensitive marker of mast tion,that a diet supplemented with n-3 PUFA can alkalize cell activation in the airways and a potent bronchocon- airway (EBC) pH in asthmatics.
strictor,and has been shown to be increased after Our finding that the PCSO-524 diet significantly decreased EBC Cyst-LT concentration supports previous studies that have shown that n-3 PUFA supplementation can attenuate Cyst-LT levels in urineand induced sputum In the present study EBC 8-isoprostanne levels were following and in EBC following dry gas hyper- reduced prior to, and following, the dry gas hyperpnea compared to a usual (normal) diet in subjects with challenge on the PCSO-524 diet compared to the placebo EIB. Studies have shown a sustained increase in Cyst-LTs and control diet.
and other bronchoconstrictve eicosanoids, such as PGD2, Oxidative damage to lipids (lipid peroxidation) leads to in the airways after an exercise and these the production of 8-isoprostane, an F2 isoprostane, formed markers have been shown to be elevated for up to 6 h after nonenzymatically by oxidation of arachidonic acid inde- an exercise challenge in asthmatic subjects with EIB.Eo- pendent of cyclooxygenase (COX) action, and considered a sinophils, mast cells and basophils can directly synthesis reliable marker of oxidative stress because it is structurally Cyst-LTs, which can cause tissue edema, stimulate airway stable and are produced in vivoLevels of 8-isoporstane in secretions, promote cell cycling and proliferation of airway EBC are increased in adultsand children with asthma, smooth muscle, and may directly increase eosinophilic and have been shown to be increased in EBC of asthmatic inflammation.It has been shown that in some patient patients with EIB, as well as correlated with the severity of populations, the amount of eosinophilia in induced sputum is correlated with EIB severity.
Although previous studies have shown that fish oil sup- The initial rate-limiting step in the formation of Cyst-LTs plementation provides a protective effect against EIB and other eicosanoids is the release of arachidonic acid and HIBas a result of its anti-inflammatory properties, from membrane phospholipids regulated by phospholipase the present study is the first to show that PCSO-524, a (PL) A2 enzymes.It has been shown that secreted phos- different type of marine oil derived from the shellfish P.
pholipase A2 group X (sPLA2-X) is elevated in induced canaliculus (NZGLM) is similarly effective as fish oil in sputum cells of asthmatic patients with and attenuating airway inflammation and HIB in asthmatic increased in the BAL fluid of asthmatics in association with lung function and eicosanoid It has been While PCSO-524 has been shown to be effective in shown that the major source of sPLA2-X is the airway treating osteoarthritis, rheumatoid arthritis, and inflam- and that the effects of exogenous sPLA2-X on matory bowel disease,only a limited number of studies human eosinophils can rapidly initiate Cyst-LT formation in have examined the efficacy of PCSO-524 in human eosinophils.These findingssuggest that sPLA2-X may and animal models of asthma.Emelyanov serve as a significant regulator of airway eicosanoid for- et al.demonstrated in 46 patients with atopic asthma mation and that this enzyme is strongly implicated in the that PCSO-524 (Lyprinol) [two capsules taken twice daily pathophysiology of EIB/HIB.
for 8 weeks; each capsule containing 50 mg n-3 PUFA and The present study has shown that PCSO-524 can 100 mg olive oil], compared to placebo, reduced daytime significantly attenuate the increased urinary levels of CC16 wheeze and exhaled hydrogen peroxide (marker of airway observed prior to, and following, dry gas hyperpnea on the inflammation) and increased morning peak expiratory flow, placebo and control diet. CC16 is a low-molecular-weight but did not improve night awakenings or reduce the use of (16 kDa) protein, secreted in large amounts into the lumen b2-agonists or forced expiratory volume in 1-sec (FEV1).
of the respiratory tract by nonciliated bronchiolar Clara Recently, Lello et studied the use of PCSO-524 cells. Following its passage into the bloodstream across the (Lyprinol) in 71 children with moderate chronic persistent air-blood barrier, CC16 is rapidly eliminated by glomerular asthma taking regular inhaled corticosteroids (ICS). These filtration. In humans, CC16 in extrapulmonary fluid has authorsfound that 16 weeks of PCSO-524 (Lyprinol) been used as indirect marker of lung epithelial cell dam- supplementation improved the percentage of children and it has been suggested that CC16 reporting slight to no problems with their asthma at 3 may play a protective role in response to epithelial stress, months of supplementation, and fewer mild and moderate such as to protect the airway from dysfunction due to asthma exacerbations overall in the PCSO-524 (Lyprinol) dehydration of the epithelium; most likely via inhibition group. Wood et assessed the effects of 14 days of fish of PLA2 and the subsequent suppression of inflammatory oil and PCSO-524 (Lyprinol) supplementation on allergic have shown that epithelial inflammation and lung function using a mouse model of stress, as shown by increases in urinary CC16 levels, occurs ovalbumin (OVA)-induced allergic airway disease (AAD).
Please cite this article in press as: Mickleborough TD, et al., Marine lipid fraction PCSO-524 (lyprinol/omega XL) of the New Zealandgreen lipped mussel attenuates hyperpnea-induced bronchoconstriction in asthma, Respiratory Medicine (2013), T.D. Mickleborough et al.
The PCSO-524 (Lyprinol) diet, but not the fish oil diet, part, why in the present study PCSO-524 was effective in reduced eosinophil influx into the bronchoalveolar lavage attenuating airway inflammation, given the very low dose fluid, lung tissue and blood, decreased mucus hypersecre- of EPA and DHA.
tion in the lung and attenuated airway hyperresponsiveness In conclusion, the present study has shown that a lipid (AHR). However, the effects seen on the PCSO-524 extract of NZGLM (PCSO-524) attenuates airway inflam- (Lyprinol) diet were not associated with changes in IgG1 mation and provides protection against HIB in asthmatic and IgG2a, or the release of the cytokines IL-4, IL-5, IL-13 studiesthat PCSO-524 may have beneficial effects in PCSO-524 is a mixture of the five main lipid classes HIB/asthma, by serving as a pro-resolving agonist and/or including sterol esters, triglycerides, free fatty acids, ste- inflammatory antagonist. Further studies are required to rols and polar PCSO-524 contains approximately determine the minimum effective dose needed to atten- 13% EPA, 21% DHA, 30% cholesterol and up to 91 fatty acid uate HIB/asthma, and to determine the bioactive compo- components such as 5,9,12,15-octodecatretraenoic acid, nents of PCSO-524 responsible for its anti-inflammatory effect in asthmatic airways.
myristic acid, palmitic acid, palmitoleic acid, stearic acid and oleic PCSO-524 has been shown to reduce thepro-inflammatory LTB4 in human monocytes,inhibit the All authors fulfilled conditions of authorship: (1) substantial formation of 5-HETE (products from the lipoxygenase contributions to conception and design of the study, pathwayattenuate the formation of LTB4 and 5-HETE acquisition of data, or analysis and interpretation of data; from human neutrophils,directly inhibit the COX-1 and (2) drafting the manuscript or revising it critically for COX-2 and to inhibit IL-1, IL-2, IL-6, TNF-a, IFN-g important intellectual content; and (3) final approval for synthesis from several LPS-stimulated isolated cell the version to be published.
In the present study the attenuation of airway inflam- mation and subsequent improvement in pulmonary function Sources of funding cannot be explained entirely by the EPA and DHA content ofPCSO-524, since the amount of EPA and DHA consumed This work was supported by a grant from Pharmalink In- daily was only 72 mg and 48 mg respectively, which is ternational Ltd, Hong Kong. The funders had no role in substantially lower than our previous studies examining the study design, data collection and analysis, in writing the effect of fish oil on EIB/HIB (3.2 g EPA/day and 2.0e2.2 g manuscript, or decision to publish.
DHA/day).Interestingly, Wood et al.have shown thatPCSO-524, but not fish oil, provides a protective effect Conflict of interest against eosinophilic inflammation, mucus production, TH2cytokine responses in the lungs and airways, and airwayhyperresponsiveness, in a murine model of AAD. In addi- The authors declare no conflicts of interest.
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Op-gbev120037 1046.1053

Weak 50-mRNA Secondary Structures in Short EukaryoticGenes Yang Ding, Premal Shah, and Joshua B. Plotkin* Department of Biology, University of Pennsylvania *Corresponding author: E-mail: [email protected]. Accepted: September 21, 2012 Experimental studies of translation have found that short genes tend to exhibit greater densities of ribosomes than long genes ineukaryotic species. It remains an open question whether the elevated ribosome density on short genes is due to faster initiation orslower elongation dynamics. Here, we address this question computationally using 50-mRNA folding energy as a proxy for translationinitiation rates and codon bias as a proxy for elongation rates. We report a significant trend toward reduced 50-secondary structure inshorter coding sequences, suggesting that short genes initiate faster during translation. We also find a trend toward higher 50-codonbias in short genes, suggesting that short genes elongate faster than long genes. Both of these trends hold across a diverse set ofeukaryotic taxa. Thus, the elevated ribosome density on short eukaryotic genes is likely caused by differential rates of initiation, ratherthan differential rates of elongation.