Treatment of asthma with lipid extract of New Zealand green-lipped mussel: a randomised clinical trial
European Respiratory Journal. Volume 20 Issue (3) Pages 596–600, 2002
A. Emelyanov1, G. Fedoseev1, O. Krasnoschekova1, A. Abulimity1, T. Trendeleva1, P.J. Barnes2
1Hospital Therapeutic Clinic, Pavlov Medical University, St-Petersburg, Russia
2Dept of Thoracic Medicine, National Heart and Lung Institute, London, UK
A. Emelyanov, Hospital Therapeutic Clinic, Pavlov Medical University, 6/8 L.Tolstogo Str, St-Petersburg, 197089, Russia,
Fax: 7 8122342749. Email: firstname.lastname@example.org
Asthma, green-lipped mussel, lipid extract, treatment.
Received: 19 December 2001, Accepted after revision 18 April 2002.
Asthma is a chronic inflammatory disease of the airways mediated, at least in part, by leukotrienes and other lipid mediators. Experimental studies have shown that lipid extract of New Zealand green-lipped mussel, Perna canaliculus, is effective in inhibiting 5´-lipoxygenase and cyclo-oxygenase pathways responsible for production of eicosanoids, including leukotrienes and prostaglandins. The aim of this study was to assess its effect on symptoms, peak expiratory flow (PEF) and hydrogen peroxide (H2O2) in expired breath condensate as a marker of airway inflammation in patients with steroid-naïve atopic asthma in a double-blind randomised, placebo-controlled clinical trial.
Forty six patients with atopic asthma received two capsules of lipid extract (Lyprinol®) or placebo b.i.d. for 8 weeks. Each capsule of lipid extract contained 50 mg v-3 polyunsaturated fatty acids and 100 mg olive oil, whereas placebo capsules contained only 150 mg olive oil.
There was a significant decrease in daytime wheeze, the concentration of exhaled H2O2 and an increase in morning PEF in the lipid extract group compared to the placebo group. There were no significant side-effects.
The authors conclude that lipid extract of New Zealand green-lipped mussel may have some beneficial effect in patients with atopic asthma.
Asthma is a chronic inflammatory disease of the airways mediated, at least in part, by leukotrienes and other lipid mediators. Experimental studies have shown that lipid extract of the New Zealand green-lipped mussel (Perna canaliculus) is effective at inhibiting 5´-lipoxygenase and cyclo-oxygenase pathways, which are responsible for the production of eicosanoids, including leukotrienes and prostaglandins [1, 2, 3]. The lipid extract Lyprinol is rich in eicosapentaenoic acid and docosahexanoic acid, v-3 fatty acids that inhibit the metabolism of arachidonic acid . This extract is effective in reducing pain, swelling and stiffness and in improving the functional index in patients with rheumatoid arthritis and osteoarthritis [5, 6]. Its efficacy in the treatment of airway inflammation is unknown.
The aim of this study is to assess the effect of lipid extract of New Zealand green-lipped mussel on symptoms, peak expiratory flow (PEF) and hydrogen peroxide (H2O2) in expired breath condensate as a marker of airway inflammation in patients with atopic asthma in a double-blind randomised, placebo-controlled clinical trial.
Volunteers aged 18]56 yrs with mild-to-moderate atopic asthma were recruited in the outpatients department of the Hospital Therapeutic Clinic, Pavlov Medical University, St-Petersburg, Russia (Table 1). Patients were diagnosed according to the American Thoracic Society definition of asthma . Diagnosis was based upon clinical history, reversibility of forced expiratory volume in one second (FEV1) of >15% and diurnal variability of PEF of >20%. Atopic status was assessed by positive skin-prick test (>3 mm) to common inhalant allergens (house-dust mite, animal allergens, pollen). All patients were sensitised to house-dust mite. Severity of asthma was classified according to the National Institutes of Health/World Health Organization (NIH/WHO) guidelines . Briefly, subjects with mild asthma had symptoms twice a week or less often with FEV1 $80% of predicted and used inhaled short-acting b2-agonists for symptom relief. Patients with moderate asthma had daily symptoms and an FEV1 between 60 and 80% pred, and used inhaled short-acting b2-agonists daily. Exclusion criteria were use of inhaled steroids within the previous 4 weeks, oral steroid treatment within the previous 3 months, current or exsmokers, clinically significant heart, renal, liver and intestinal disorders, women of childbearing potential not using adequate contraception. Only patients fully cooperative in all procedures were considered for inclusion in the run-in period of the trial.
The study was approved by the Local Ethics Committee and all participants gave written consent.
The study was a single-centre, double-blind, parallel-group, randomised, placebo-controlled trial. Patients who fulfilled the inclusion criteria at the initial visit entered a 2 week observation run-in period, in which regular treatment was stopped and short-acting b2-agonists (salbutamol or terbutaline) as required were the only medications allowed. Asthma symptoms and use of rescue medications were recorded in a diary. Patients were excluded if they received inhaled steroids or were hospitalised for asthma during the run-in period.
After the run-in period patients were randomly assigned to receive either lipid extract of New Zealand green-lipped mussel, two capsules twice daily or matching placebo for a period of 8 weeks. Each lipid extract capsule contained 50 mg v-3 polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) and 100 mg olive oil (Lyprinol®, Mac Lab, Melbourne, Australia). Each identical placebo capsule contained 150 mg olive oil only. Randomisation was computer-generated in balanced blocks of two treatment regimens. Inhaled salbutamol delivered via pressurised metered-dose inhaler or terbutaline delivered by dry powder inhaler (Turbuhaler) were used as rescue medications throughout the study. No other medications were allowed. All short-acting inhaled b2-agonists were stopped at least 6 h before baseline FEV1 and exhaled H2O2 measurements.
Subjects kept a twice-daily dairy card throughout the study. They were provided with individual peak-flow meters (Vitalograph Ltd, Buckingham, UK). The best of three peak flow values was recorded before taking the morning and evening study medications. Patients recorded daytime wheeze, nocturnal awakenings from asthma, daily use of short-acting inhaled b2-agonists and compliance with the study medications. Asthma symptoms were ranked on a scale from 0 (none) to 3 (very uncomfortable and interferes with normal activities). At the beginning and the end of the study, subjects attended the research clinic for measurement of FEV1, which was measured by dry spirometry (Vitalograph Ltd). The concentration of exhaled H2O2 was measured at the beginning of the study, and at 4 and 8 weeks of treatment. Study medications and inhaled short-acting b2-agonists were withheld for at least 6 h before spirometry and exhaled H2O2 measurements.
Expired breath condensate was collected using a glass condensing device, which was placed in a large chamber with ice as previously described . After mouth rinsing, subjects breathed tidally through a mouthpiece for 20 min while wearing a nose-clip. The mouthpiece was also used as a saliva trap. The volume of condensate was 2]4 mL and H2O2 was assayed immediately. H2O2 was measured by using a colorimetric assay as described previously . Briefly, 100 µL of condensate was mixed with 100 µL of 3,3´,5,5´ tetramethylbenzidine in 0.42 M citrate buffer, pH 3.8, and 10 µL of horseradish peroxidase (52.5 U·m-1). The samples were incubated at room temperature for 20 min and the reaction stopped by the addition of 10 µL 18 N sulphuric acid. The reaction product was measured spectrophotometrically (Model 46; Lomo Inc., St-Petersburg, Russia) at 450 nm. A standard curve of H2O2 was performed for each assay.
Safety of the treatment
At each visit subjects were specially asked for any adverse events and health problems that may have occurred. All unusual signs and symptoms were recorded for further consideration. The safety of the treatment was evaluated by monitoring blood pressure, concentrations of creatinine, bilirubin, activity of liver transaminases and alkaline phosphatase in serum at clinical visits.
Analysis of data
Paired and unpaired two-tailed t-tests were used for statistical analysis. A p-value <0.05 was considered significant, and the data are expressed as mean±SEM.
Of 60 volunteers recruited to the study, 46 fulfilled the entry criteria and were randomised to treatment. The most common reason for withdrawal before randomisation was deterioration in asthma control (14 subjects). These patients had a lower FEV1 (70.3±2.1% pred) than those who were randomised (87.60±2.5, p<0.001). All randomised patients completed the treatment and were included in the statistical analysis. Thirty six of them had mild asthma and 10 patients had moderate asthma.
The mean self-recorded compliance with the two-times daily study medications (Lyprinol or placebo) was 95% and 93%, respectively.
Asthma symptoms and bronchodilator use
Mean daytime wheeze was significantly reduced in patients treated with Lyprinol compared to those treated with placebo (Table 2). There were no significant differences between the two treatment groups in respect to nocturnal awakening and use of short-acting b2-agonists (Table 2).
Lung function tests
Mean FEV1 and evening PEF during the treatment with Lyprinol did not significantly differ from those during the treatment with placebo. However, mean morning PEF was significantly higher during the treatment with Lyprinol than with placebo (Table 2).
Exhaled hydrogen peroxide
Mean concentrations of exhaled H2O2 were significantly reduced during the treatment with Lyprinol compared to placebo (Table 2).
Safety of the treatment
One patient treated with Lyprinol and one patient treated with placebo had skin itch. One patient in the Lyprinol group and two patients in the placebo group complained of a metallic taste. There were no significant changes in blood pressure, or in concentrations of serum creatinine, bilirubin, liver transaminases or alkaline phosphatase in either group of patients (data not shown).
The data from this study show that a stabilised lipid extract of New Zealand green-lipped mussel, Perna canaliculus (Lyprinol), reduces daytime wheeze and improves morning PEF in steroid-naïve patients with atopic asthma compared to placebo. There were no differences between patients treated with Lyprinol and placebo with respect to FEV1. It should be noted that most of the patients had mild asthma and FEV1 >80% pred and thus there was little room for improvement in this measurement.
Lyprinol was well tolerated and had very few side-effects. The findings of the present study are in agreement with a previously published study .
The mechanism by which lipid extract of New Zealand green-lipped mussels exerts its beneficial effect remains to be elucidated. Lyprinol inhibits leukotriene production by stimulated human polymorphonuclear leukocytes in vitro . Much of this activity was associated with its content of v-3 polyunsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) and antioxidants such as carotenoids. v-3 polyunsaturated acids inhibit arachidonate metabolism by 5´-lipoxygenase and cyclo-oxygenase , thus reducing the formation of leukotrienes and prostaglandins. Dietary supplementation with v-3 polyunsaturated fatty acids can suppress synthesis of interleukin-1 and tumour necrosis factor-a by mononuclear cells .Epidemiological studies in Inuit, Dutch , American , Australian  and Japanese populations  have shown that a high dietary intake of v-3 polyunsaturated fatty acids is associated with lower incidence of cardiovascular disease and inflammatory diseases, such as asthma and rheumatoid arthritis. Dietary supplementation with fish oil rich in the v-3 polyunsaturated fatty acids over a 10-month period is beneficial in children with asthma following a strictly controlled diet and controlled allergen exposure .
Leukotrienes are mediators of airway inflammation in asthma. They induce bronchoconstriction and airway smooth muscle proliferation [17, 18], stimulate mucus secretion , increase microvascular permeability and cause infiltration of inflammatory cells such as eosinophils and neutrophils into the airways [20, 21]. These cells release superoxide anions which then undergo spontaneous or enzyme-catalysed dismutation to form H2O2. H2O2 is a highly reactive oxygen species involved in cellular injury via further reactions leading to hydroxyl radical and lipid peroxidation products . Increased production of free radicals occurs in airway inflammation, and H2O2 is detectable in exhaled air .
Several studies have shown that H2O2 in expired breath condensate may be a nondirect marker of oxidative stress and airway inflammation in asthmatic patients [24, 25]. Exhaled H2O2 levels have previously been related to the eosinophil differential counts in induced sputum and activity of peripheral neutrophils in asthmatic patients [25, 26]. Therefore, elevated concentrations of H2O2 may result from an enhanced number and activity of inflammatory cells in the airways.
In the present study, the authors found that elevated concentrations of exhaled H2O2 were significantly decreased during treatment with Lyprinol compared to placebo. This may result from a reduction in the number of inflammatory cells in asthmatic airways because of decreased synthesis of leukotrienes and may also be due to the antioxidants such as b-carotene contained in Lyprinol.
In conclusion, this study shows that a lipid extract of New Zealand green-lipped mussel may have some beneficial effects in patients with steroid-naïve atopic asthma. Further clinical studies are needed to determine whether it may be useful as a supplement to other medications in the treatment of asthmatic patients.
1. Whitehouse MW, Macrides TA, Kalfatis NK, Betts WH, Haynes DR, Broadbent J. Anti-inflammatory activity of a lipid fraction (Lyprinol) from the NZ green-lipped mussel Inflammopharmacology 1997 Vol. 5 pp. 237–246
2. Rainsford KD, Whitehouse MW. Gastroprotective and anti-inflammatory properties of green-lipped mussel (Perna canaliculus) preparation Arzn Forsch 1980 Vol. 30 pp. 2128–2132
3. Halpern G-M. Anti-inflammatory effects of a stabilized lipid extract of Perna Canaliculus (Lyprinol®) Allerg Immunol (Paris) 2000 Vol. 32 pp. 272–278
4. Sinclair AJ, Murphy KJ, Li D. Marine lipids: overview “news insights and lipid composition of Lyprinol” Allerg Immunol (Paris) 2000 Vol. 32 pp. 261–271
5. Gibson SLM, Gibson RG. The treatment of arthritis with lipid extract of Perna canaliculus: a randomized trial Compl Ther Med 1998 Vol. 6 pp. 122–126
6. Gibson RG, Gibson S, Conway V, Chappel D. Perna canaliculus in the treatment of arthritis Practitioner 1980 Vol. 224 pp. 955–960
7. American Thoracic Society. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma Am Rev Respir Dis 1987 Vol. 136 pp. 225–244
8. Diagnosis and classificationIn: Global Initiative for asthma. NHLB/WHO Workshop Report. Publication number 95]3659USA, National Heart Lung Blood Institute, 1995; pp. pp. 48–61.
9. Emelyanov A, Fedoseev G, Abulimity A. et al. Elevated concentrations of exhaled hydrogen peroxide in asthmatic patients Chest 2001 Vol. 120 pp. 1136–1139
10. Gallati H, Pracht I. Horseradish peroxidase: kinetic studies and optimisation of peroxide activity determination using the substrates H2O2 and 3,3´,5,5´-tetramethybenzidine J Clin Chem Clin Biochem 1985 Vol. 23 pp. 453–460
11. Endres S, Ghorbani R, Kelly VE. et al. The effect of dietary supplementation with v-3 polyunsaturated fatty acids on the synthesis of interleukin-1 and tumor necrosis factor by mononuclear cells N Engl J Med 1989 Vol. 320 pp. 265–271
12. Kromann N, Green A. Epidemiological studies in the Upernavik district, Greenland. Incidence of some chronic diseases 1950]1974 Acta Med Scan 1980 Vol. 208 pp. 401–406
13. Schwartz J, Weiss ST. The relationship of dietary fish oil intake to level of pulmonary function in the first National Health and Nutritional Survey Eur Respir J 1994 Vol. 7 pp. 1821–1824
14. Peat JK, Salome CM, Woolcock AJ. Factors associated with bronchial hyperresponsiveness in Australian adults and children Eur Respir J 1992 Vol. 5 pp. 921–929
15. Satomi H, Minowa M, Hatano S, Iikura Y. An epidemiological study of the preventive effect of dietary fish on bronchial asthma Bull Inst Public Health 1994 Vol. 43 pp. 305–314
16. Nagakura T, Matsuda S, Shichijyo K, Sugimoto H, Hata K. Dietary supplementation with fish oil rich in v-3 polyunsaturated fatty acids in children with bronchial asthma Eur Respir J 2000 Vol. 16 pp. 861–865
17. Piper PJ. Leukotrienes: potent mediators of airway constriction Int Arch Allergy Appl Immunol 1985 Vol. 76 Suppl. 1, pp. 43–48
18. Wang CG, Du T, Xu LJ, Martin JG. Role of leukotriene D4 in allergen-induced increases in airway smooth muscle in rat Am Rev Respir Dis 1994 Vol. 148 pp. 413–417
19. Marom Z, Shelhamer JH, Bach MK, Morton DR, Kaliner M. Slow-reacting substances leukotriene C4 and D4, increase the release of mucus from human airway in vitro Am Rev Respir Dis 1982 Vol. 126 pp. 449–451
20. Dahlen S-E, Bjorck, Heaqvist P. et al. Leukotrienes promote plasma leakage and leukocyte adhesion in post-capillary venules: in vitro effects with relevance to the acute inflammatory response Proc Natl Acad Sci USA 1981 Vol. 78 pp. 3887–3891
21. Laitinen LA, Laitinen A, Haahtela T, Vilkka V, Spur BW, Lee TH. Leukotriene E4 cause granulocyte infiltration into asthmatic airway Lancet 1993 Vol. 341 pp. 989–990
22. Mates JM, Sanchez-Jimenez F. Antioxidant enzymes and their implications in pathophysiologic processes Front Biosci 1999 Vol. 4 pp. 339–345
23. Kharitonov SA, Barnes PJ. Exhaled markers of pulmonary diseases Am J Respir Crit Care Med 2001 Vol. 163 pp. 1693–1722
24. Antzak A, Nowak D, Shariati B, Krol M, Piasecka G, Kurmanowska Z. Increased hydrogen peroxide and thiobarbituric acid-reactive products in expired breath condensate of asthmatic patients Eur Respir J 1997 Vol. 9 pp. 1235–1241
25. Horvath I, Donnely LE, Kiss A. et al. Combined use of exhaled hydrogen peroxide and nitric oxide in monitoring asthma Am J Respir Crit Care Med 1998 Vol. 158 pp. 1042–1046
26. Antzak A, Nowak D, Bialasiewicz P, Kasielski M. Hydrogen peroxide in expired air condensate correlates positively with early steps of peripheral neutrophil activation in asthmatic patients Arch Immunol Ther Exp (Warsz) 1999 Vol. 47 pp. 119–126