134
Views
20
CrossRef citations to date
0
Altmetric
Clinical Trial Report

The effect of conjugated linoleic acid on oxidative stress and matrix metalloproteinases 2 and 9 in patients with COPD

, , , &
Pages 1449-1454 | Published online: 03 May 2018

Abstract

Background

Natural antioxidants in foods may be used in prevention and treatment of oxidative stress and inflammation in COPD. Therefore, this study aimed to evaluate the effect of conjugated linoleic acid (CLA) supplement as natural antioxidants on oxidative stress levels, and MMP2 and MMP9 serum levels in COPD patients.

Materials and methods

This clinical trial study was conducted on 90 (supplement group=45 and control group=45) COPD patients in Ardabil city, Iran, in 2015. After obtaining written consent, general information was collected from each patient using a validated and reliable questionnaire. Supplement group received 3.2 g of CLA and those in the control group were given 3.2 g of placebo for 6 weeks on a daily basis. Fasting blood samples were taken from all of the patients for testing of malondialdehyde (MDA), MMP2, and MMP9 levels at the beginning and end of the study. Data were analyzed using Kolmogorov–Smirnov test, independent samples t-test, paired sample t-test, chi-square test, and ANOVA.

Results

There were no significant differences between the two groups with regard to mean age, smoking status, and serum level of MDA at the beginning of the study. In the supplement group, the serum level of MDA decreased significantly at the end of the 6th week compared to that in the beginning of the study (p=0.0004), while in the placebo group, the difference was found to be insignificant. The serum level of MMP9 decreased significantly in the supplement group, while in the placebo group its level increased significantly as compared to that at the beginning of the study (p<0.05). The serum levels of MMP2 indicated no significant differences between the two groups neither at the beginning nor at the end of the study.

Conclusion

These findings indicated that CLA supplementation may be helpful for COPD patients through inhibiting the production of oxidative stress and controlling MMP9 serum levels.

Introduction

COPD is the most prevalent chronic respiratory disease and one of the main causes of death throughout the world. It is characterized by progressive and irreversible airflow obstruction.Citation1 From among the mechanisms involved in the pathogenesis of this disease, oxidative stress and inflammation have attracted increasing attention.Citation2 Oxidative stress is indicative of an increase in the production of various types of free radicals of oxygen from inflammatory cells and the inability of the biological system to detoxify them or repair their damaging effects, thus resulting in oxidative damage to cells, tissues, and organs of the body.Citation3 The increase of oxidative stress in lungs causes an increase in the transcription of inflammatory genes, protease activity, mucus secretion, and resistance to glucocorticoid.Citation4 An increase in the level of free radicals of oxygen leads to an augment, either directly or through lipid peroxidation products, in the inflammatory response, which has now been recognized as the main pathogenic factor in the progression of COPD and the induction of matrix metalloproteinase (MMP) expressions.Citation5 MMPs are a large family of protease enzymes, and 26 types have been recognized to date. It seems that MMPs have the most important role in the destruction and synthesis of collagen-rich tissues in different physiological and pathological conditions.Citation6 Among the enzymes in MMP family, the two enzymes of MMP2 (72 kDa gelatinase) and MMP9 (92 kDa gelatinase) have highest activity. MMP9 exists in low levels in the lung tissues of healthy humans. But, its level increases in severe pulmonary diseases such as asthma, primary pulmonary fibrosis, COPD, and cystic fibrosis leading to an increase in disease severity and resistance to treatment.Citation7Citation9 In a study, an increase in the level of MMP2 was observed in the sputum samples taken from patients with asthma and COPD, which was statistically significant compared to the patients in control group.Citation10 The destruction of the elasticity of the alveolar wall in COPD and its false reconstruction due to increased proteolytic activity result in the loss of alveolar function and disease progression. For this reason, MMPs have nowadays attracted increasing attention since they can have a potential role in the destruction and reconstruction of damaged alveoli.Citation11 Regarding the role of oxidative stress in the pathogenesis of COPD, the use of antioxidant drugs as a new strategy for the treatment and prevention of this disease has become important.Citation12 One of the supplements with antioxidant properties that has recently been focused on by many researchers due to its beneficial effects on human health condition is conjugated linoleic acid (CLA), which can be used together with usual treatments.Citation12 CLA is a fatty acid with multiple double bonds which naturally exists in animal resources.Citation13 The CLA supplements with different isomers can be used as antioxidant, anti-inflammatory, anticancer, and anti-atherosclerosis substances; they can also be used as a stimulant of immune system and growth factor, for changing of body composition, and so on.Citation14,Citation15 Although a number of studies have demonstrated possible benefits of CLA intake on inflammatory factors,Citation16,Citation17 there is not enough evidence to establish positive effects of CLA consumption in reducing oxidative stress and inflammatory factors in the COPD subjects. There are no studies addressing the effect of CLA supplement on the levels of oxidative stress indices and serum levels of MMPs in COPD patients. Therefore, this study aimed to evaluate the effect of CLA supplement on the levels of oxidative stress, MMP2, and MMP9 in COPD patients.

Materials and methods

This study was a double-blind clinical trial which was conducted on COPD patients in the Pulmonary Department at Imam Khomeini Hospital, Ardabil city, Iran, in 2015. At the beginning of the study, 168 male patients within the age range of 40–80 years were selected based on the criteria proposed by American Thoracic Society for having COPD, that is, coughing, phlegm production, and chronic dyspnea together with spirometry results with forced expiratory volume in first second (FEV1)/forced vital capacity (FVC) ratio below 70%. Seventy-five patients, who were suffering from other chronic diseases such as cardiovascular diseases, diabetes, malignancies, etc, were excluded. Three patients were excluded during the study due to lack of cooperation and abuse of CLA supplements. Written informed consent form was obtained from each patient before the study. In order to collect the needed information about the patients such as their name, surname, age, education level, job, place of residence, smoking history and abandonment, and the number of exacerbations of disease during the previous year, a validated and reliable questionnaire was used for each patient. Finally, 90 patients (supplement group=45 and control group=45) were evaluated. This research was ethically approved in the research department of Ardabil University of Medical Sciences (Iran) with the code of “Arums.REC.93.56” and recorded in the Clinical Trial Center of Iran with the code of IRCT2015080823559N1.

Supplementation

The patients in the supplement group received 3.2 g of CLA in softgel capsules (with 80% purity), which include c9-t11 and c12-t10 isomers (50/50) and are manufactured in the form of tablets in the US with Nutrex research company the commercial name of Nutrex (Nutrex Reaserch Inc., Oviedo, FL, USA), while the control group received the same amount of placebo for 6 weeks. Before the beginning of the study, the containers of the CLA supplement and placebo were coded with letters A and B, respectively, to make sure that neither the interviewers nor the patients knew which group received which type of supplement, and therefore the process was double-blind.

Biochemical information

At the beginning of the study and at the end of the 6th week, 3 mL of venous blood samples were taken from all of the patients after 12 hours of fasting and gently transferred to test tubes. Then, the samples were labeled and transferred to the laboratory. After that, they were centrifuged for 10 minutes at 4,000 rpm to separate the serum. Finally, the samples were distributed in four micro-tubes and kept at the temperature of −80°C for malondialdehyde (MDA), MMP2, and MMP9 tests.

The serum level of MDA was assessed by thiobarbituric acid reactive substances test using the reaction of thiobarbituric acid. MDA is one of the oxidative stress biomarkers,Citation18 and it has been shown that there is an association between serum levels and COPD severity.Citation19,Citation20 The serum samples of patients were first heated with thiobarbituric acid under acidic conditions according to the instructions provided by the kit manufacturer and then cooled down. Afterward, the resulting color combination in n-butanol was extracted. The absorbance of the pink color was measured at 530 nm. Serum MMP2 and MMP9 levels were assayed by standard enzyme-linked immunosorbent assay (ELISA) kits (Boster, Wuhan, China). Serum samples were diluted 1:100 according to the manufacturer’s specifications for MMP9 and MMP2 assay. For the accuracy of assessment, the experiments were performed in duplicate. The colors formed were read at the wavelengths of 450 and 630 nm by Anhos 2000 microplate reader, and the sample concentrations were calculated using Multcalc Software (Wallac, Turku, Finland).

Patients’ follow-up

In order to both control the consumption of supplementation and prevent the loss of samples, the follow-up was also done through face-to-face visits, in addition to making telephone calls. The pills needed for 6 weeks were given to the patients. They were asked to return the remaining pills if they stopped consuming them. In the following visits, 97% of the patients reported to have taken their pills regularly and were studied to find out the effects of the supplement on their disease. The compliance of patients in terms of taking their supplements was evaluated based on the number of remaining pills at the end of the 6th week of the study. The researcher provided the patients with some information about the possible complications of the supplement and gave them his phone number to contact him if they encountered any problem.

Data analysis and statistical methods

In order to check the normality of the data related to each variable, Kolmogorov–Smirnov test was employed. The information in the checklists was then transferred to SPSS Software (version 16), and in order to find the relationship between different variables, independent samples t-test, paired sample t-test, chi-square test, and ANOVA were performed. The significance level was taken to be 0.05 for all the tests. The results were reported in the form of mean ± standard deviation.

Results

Demographic information and risk factors related to the patients and their comparison between the two groups are presented in and . Most of the patients in this study were urban residents. Most of the patients were farmers, and most of the subjects were illiterate. Based on the chi-square test, there was no significant difference with regard to variables (such as FEV1 percentage, age, education level, occupational levels, smoking history and abandonment, and the number of exacerbations) between the two groups. The mean age of supplement and placebo groups was 63.60±10.94 (mean ± SD) years and 61.64±10.60 years, respectively. The results indicated that there was no significant difference between the two groups regarding serum level of MDA at the beginning and end of the study. However, paired sample t-test revealed that the serum level of MDA decreased significantly at the end of the 6th week compared to that at the beginning of the study (p=0.0004) in the supplement group, while in the placebo group, the difference was found to be insignificant (p=0.3; ). There were no significant differences in the serum levels of MMP2 between the two groups at the beginning and end of the study. The serum level of MMP2 decreased and increased insignificantly in supplement and placebo groups, respectively (). The results of the analysis of MMP9 levels revealed that at the beginning of the study, the two groups were not significantly different in this regard. However, the difference turned out to be significant at the end of the study (p<0.05). Paired sample t-test indicated that at the end of the 6th week, the serum level of MMP9 decreased significantly in the supplement group, while in the placebo group, its level increased significantly as compared to the beginning of the study (p<0.05; ).

Table 1 Demographic information of the COPD patients (n=90)

Table 2 Risk factors related to the COPD patients and their comparison between the two groups

Table 3 The average level of MDA in the supplement and control groups

Table 4 The average serum levels of MMP2 and MMP9 in the supplement and placebo groups

Discussion

The results of this study indicated that CLA significantly decreased the serum levels of MDA and MMP9 in COPD patients at the end of the 6th week. In the placebo group, the serum levels of MMP9 increased significantly in patients. In the COPD patients, an increase in the level of oxidative stress has been observed, which might play a role in the pathogenesis of this disease.Citation3 The increase of oxidative stress and different types of serum MMPs, especially MMP2 and MMP9, has been reported as important factors in the pathogenesis of COPD in the patients suffering from this disease.Citation3 In COPD, body cells are constantly exposed to various oxidizing agents. Reactive oxygen species (ROS) is one of the oxidizing compounds produced in human body.Citation3,Citation21 On the other hand, analyses in laboratory conditions have shown that the increase of oxidative stress acts as a trigger for the increase of MMPs, especially MMP2 and MMP9.Citation11 Therefore, controlling oxidative stress and inhibiting the synthesis of MMPs are important in the treatment of this disease and can be considered as a new therapeutic strategy.Citation22 Although there are few studies about the effect of CLA on different cancers and some chronic diseases,Citation15,Citation23 very limited investigations have been conducted about the effect of this supplement on COPD patients.

The results of this study are consistent with the findings of the studies conducted by Baghi et al on healthy males,Citation16 Hassan Eftekhari et al on cardiovascular patients,Citation24 and Aryaeian et al on patients with rheumatoid arthritis.Citation25 In their studies, they indicated that the consumption of CLA supplement can decrease the serum level of MDA, which is an index of oxidative stress. The reason for the decrease in the serum level of MDA might be the anti-inflammatory effect of CLA as the result of the activation of peroxisome proliferator-activated receptors. Through this process, CLA finally leads to the inhibition of some key factors involved in the induction of oxidative stress and triggers the production of some proinflammatory factors.Citation26Citation28 During the present study, the serum level of MDA significantly decreased in the supplement group, while in the placebo group, its level slightly increased. Antioxidant enzymes and compounds are produced in the body to cope with oxidative stress conditionsCitation21 so as to protect the cells and living tissues of the body against the damages caused by free radicals and ROS.Citation3 Therefore, treatment with nutritional antioxidants might be helpful. In other study, Shadman et al found that the consumption of CLA did not change the serum level of MDA significantly in patients with type II diabetes.Citation29 Their finding is inconsistent with the results of our study, the possible reasons for which might be the different statistical populations addressed in each study and the increase of resistance to insulin and peroxidation lipids as the result of CLA consumption in patients with diabetes.Citation29

In the present study, the serum concentration of MMP9 significantly decreased in COPD patients after the consumption of CLA, which shows the positive effect of this supplement. Our results are in line with the findings of the studies conducted by Hubbard et al,Citation30 Harris et al,Citation31 and Soel et al,Citation32 who found that the consumption of CLA can decrease the serum level of MMP9 in healthy and cancerous rats. Although the exact mechanism of action of CLA on MMP2 and MMP9 in COPD patients is unknown, some researchers have reported that it can affect the gene expression of MMP2 and MMP9 and suppress their activity via stopping the production of mRNAs.Citation30 An evidence for this explanation can come from the study of Hubbard et al in which they observed the destruction of mRNAs of these MMPs in rats.Citation30 Another mechanism may be related to the regulation of gene expression of MMPs by cytokines.Citation22 Probably, the consumption of CLA decreases the production of MMPs via the inhibition of cytokines.Citation33 Finally, the third mechanism of action is the decrease in the production of oxidative stress considering the fact that MMPs alone are inactive and it is the oxidative stress that activates them.Citation6 Platt et al have shownCitation34 that the CLA did not affect the level of MMP9, which is inconsistent with our findings. This inconsistency might be due to the different settings in which the study has been conducted and also the individual differences of the patients under study, such as age, gender, and having other diseases, and the duration of the intervention should also be considered.Citation11

The duration of intervention and assessment of CLA blood levels in the patients were the main limitations of this study.

Conclusion

The serum levels of MDA and MMP9 decreased signifi-cantly, and an insignificant decrease in the serum level of MMP2 at the end of the 6th week after CLA supplementation was observed. These findings indicated that CLA supplementation may be helpful for COPD patients through inhibiting the production of oxidative stress and controlling MMP9 serum levels.

Acknowledgments

We are grateful to Ardabil University of Medical Sciences for financial support, to all the subjects for their participation, and to the Vice-Chancellor’s Office for allowing us to conduct this study at Ardabil University of Medical Sciences and for help in data collection.

Disclosure

The authors report no conflicts of interest in this work.

References

  • PauwelsRARabeKFBurden and clinical features of chronic obstructive pulmonary disease (COPD)Lancet2004364943461362015313363
  • RahmanIThe role of oxidative stress in the pathogenesis of COPD: implications for therapyTreat Rspir Med200543175200
  • LipinskiBHydroxyl radical and its scavengers in health and diseaseOxid Med Cell Longev2011201180969621904647
  • BowlerRPBarnesPJCrapoJDThe role of oxidative stress in chronic obstructive pulmonary diseaseCOPD20041225527717136992
  • KirkhamPRahmanIOxidative stress in asthma and COPD: antioxidants as a therapeutic strategyPharmacol Ther2006111247649416458359
  • ChurgAZhouSWrightJLSeries “matrix metalloproteinases in lung health and disease”: matrix metalloproteinases in COPDEur Respir J201239119720921920892
  • MercerPFShuteJKBhowmikADonaldsonGCWedzichaJAWarnerJAMMP-9, TIMP-1 and inflammatory cells in sputum from COPD patients during exacerbationRespir Res20056115115516372907
  • NavratilovaZKolekVPetrekMMatrix metalloproteinases and their inhibitors in chronic obstructive pulmonary diseaseArch Immunol Therap Exp2016643177193
  • BrajerBBatura-GabryelHNowickaAKuznar-KaminskaBSzczepanikAConcentration of matrix metalloproteinase-9 in serum of patients with chronic obstructive pulmonary disease and a degree of airway obstruction and disease progressionJ Physiol Pharmacol200859Suppl 6145152
  • Agha-HosseiniFMirzaii-DizgahIFarmanbarNAbdollahiMOxida-tive stress status and DNA damage in saliva of human subjects with oral lichen planus and oral squamous cell carcinomaJ Oral Pathol Med2012411073674022582895
  • BelvisiMGBottomleyKMThe role of matrix metalloproteinases (MMPs) in the pathophysiology of chronic obstructive pulmonary disease (COPD): a therapeutic role for inhibitors of MMPs?Inflamm Res20035239510012755372
  • BhattacharyaABanuJRahmanMCauseyJFernandesGBiological effects of conjugated linoleic acids in health and diseaseJ Nutr Biochem2006171278981016650752
  • IngSWBeluryMAImpact of conjugated linoleic acid on bone physiology: proposed mechanism involving inhibition of adipogenesisNutr Rev201169312313121348876
  • RaffMTholstrupTBasuSNonboePSorensenMTStraarupEMA diet rich in conjugated linoleic acid and butter increases lipid per-oxidation but does not affect atherosclerotic, inflammatory, or diabetic risk markers in healthy young menJ Nutr2008138350951418287358
  • TholstrupTRaffMStraarupEMLundPBasuSBruunJMAn oil mixture with trans-10, cis-12 conjugated linoleic acid increases markers of inflammation and in vivo lipid peroxidation compared with cis-9, trans-11 conjugated linoleic acid in postmenopausal womenJ Nutr200813881445145118641189
  • BaghiANMazaniMNematiAAmaniMAlamolhodaSMogadamRAAnti-inflammatory effects of conjugated linoleic acid on young athletic malesJPMA J Pak Med Assoc201666328028426968277
  • OraldiMMaggioraMPaiuzziECanutoRAMuzioGCLA reduces inflammatory mediators from A427 human lung cancer cells and A427 conditioned medium promotes differentiation of C2C12 murine muscle cellsLipids2013481293823129255
  • KhoubnasabjafariMAnsarinKJouybanAReliability of malondialdehyde as a biomarker of oxidative stress in psychological disordersBioImpacts20155312326457249
  • BajpaiJPrakashVKantSStudy of oxidative stress biomarkers in chronic obstructive pulmonary disease and their correlation with disease severity in north Indian population cohortLung India201734432428671162
  • ZengMLiYJiangYLuGHuangXGuanKLocal and systemic oxidative stress status in chronic obstructive pulmonary disease patientsCan Respir J2013201354123457673
  • MittlerROxidative stress, antioxidants and stress toleranceTrends in Plant Science20027940541012234732
  • RoomiMWMonterreyJCKalinovskyTRathMNiedzwieckiAPatterns of MMP-2 and MMP-9 expression in human cancer cell linesOncol Rep20092151323133319360311
  • YangMCookMEDietary conjugated linoleic acid decreased cachexia, macrophage tumor necrosis factor-alpha production, and modifies splenocyte cytokines productionExp Biol Med200322815158
  • Hassan EftekhariMAliasghariFBabaei-BeigiMAHasanzadehJEffect of conjugated linoleic acid and omega-3 fatty acid supplementation on inflammatory and oxidative stress markers in atherosclerotic patientsARYA Atheroscler20139631131824575132
  • AryaeianNDjalaliMShahramFDjazayeryAEshragianMREffect of conjugated linoleic acid, vitamin E, alone or combined on immunity and inflammatory parameters in adults with active rheumatoid arthritis: a randomized controlled trialInt J Prev Med20145121567157725709792
  • ButzDELiGHuebnerSMCookMEA mechanistic approach to understanding conjugated linoleic acid’s role in inflammation using murine models of rheumatoid arthritisAm J Physiol Regul Integr Comp Physiol20072932R669R67617553852
  • MohammadzadehMFaramarziEMahdaviRNasirimotlaghBAsghari JafarabadiMEffect of conjugated linoleic acid supplementation on inflammatory factors and matrix metalloproteinase enzymes in rectal cancer patients undergoing chemoradiotherapyIntegr Cancer Ther201312649650223632235
  • ParkNYValacchiGLimYEffect of dietary conjugated linoleic acid supplementation on early inflammatory responses during cutaneous wound healingMediators Inflamm20102010 Pii: 342328
  • ShadmanZTalebanFASaadatNHedayatiMEffect of conjugated linoleic acid and vitamin E on glycemic control, body composition, and inflammatory markers in overweight type2 diabeticsJ Diabetes Metab Disord20131214223870044
  • HubbardNELimDEricksonKLConjugated linoleic acid alters matrix metalloproteinases of metastatic mouse mammary tumor cellsJ Nutr200713761423142917513401
  • HarrisMAHansenRAVidsudhiphanPEffects of conjugated linoleic acids and docosahexaenoic acid on rat liver and reproductive tissue fatty acids, prostaglandins and matrix metalloproteinase productionProstaglandins Leukot Essent Fatty Acids2001651232911487304
  • SoelSMChoiOSBangMHYoon ParkJHKimWKInfluence of conjugated linoleic acid isomers on the metastasis of colon cancer cells in vitro and in vivoJ Nutr Biochem2007181065065717368880
  • GhobadiHMatinSNematiANaghizadeh-BaghiAThe effect of conjugated linoleic acid supplementation on the nutritional status of COPD patientsInt J Chron Obstruct Pulmon Dis2016112711272027843307
  • PlattIEl-SohemyAEffects of 9 cis, 11 trans and 10 trans, 12 cis CLA on osteoclast formation and activity from human CD14+ monocytesLipids Health Dis2009811519402897