This article was downloaded by: [New York University] On: 24 June 2015, At: 07:01 Publisher: Routledge Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of the American College of Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uacn20
Effects of Probiotic Supplementation on Oxidative Stress Indices in Women with Rheumatoid Arthritis: A Randomized Double-Blind Clinical Trial a
b
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Elnaz Vaghef-Mehrabany MS , Aziz Homayouni-Rad PhD , Beitullah Alipour PhD , Sakinehd
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e
Khatoun Sharif PhD , Leila Vaghef-Mehrabany MS & Serour Alipour-Ajiry MS a
Department of Nutrition, Biochemistry and Diet Therapy, Tabriz University of Medical Sciences, Tabriz, IRAN b
Department of Food Science and Technology, Tabriz University of Medical Sciences, Tabriz, IRAN
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c
Department of Community Nutrition, Tabriz University of Medical Sciences, Tabriz, IRAN
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Rheumatology Department, Tabriz University of Medical Sciences, Tabriz, IRAN
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Faculty of Nutrition, Research Vice Chancellor, Tabriz University of Medical Sciences, Tabriz, IRAN f
Department of Clinical Nutrition, Faculty of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, Tabriz, IRAN Published online: 09 Apr 2015.
To cite this article: Elnaz Vaghef-Mehrabany MS, Aziz Homayouni-Rad PhD, Beitullah Alipour PhD, Sakineh-Khatoun Sharif PhD, Leila Vaghef-Mehrabany MS & Serour Alipour-Ajiry MS (2015): Effects of Probiotic Supplementation on Oxidative Stress Indices in Women with Rheumatoid Arthritis: A Randomized Double-Blind Clinical Trial, Journal of the American College of Nutrition, DOI: 10.1080/07315724.2014.959208 To link to this article: http://dx.doi.org/10.1080/07315724.2014.959208
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Original Research
Effects of Probiotic Supplementation on Oxidative Stress Indices in Women with Rheumatoid Arthritis: A Randomized Double-Blind Clinical Trial Elnaz Vaghef-Mehrabany, MS, Aziz Homayouni-Rad, PhD, Beitullah Alipour, PhD, Sakineh-Khatoun Sharif, PhD, Leila Vaghef-Mehrabany, MS, and Serour Alipour-Ajiry, MS
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Department of Nutrition, Biochemistry and Diet Therapy (E.V.-M.), Department of Food Science and Technology (A.H.-R.), Department of Community Nutrition (B.A.), Rheumatology Department (S.-K.S.), Faculty of Nutrition, Research Vice Chancellor (S.A.-A.), Tabriz University of Medical Sciences, Tabriz, IRAN; Department of Clinical Nutrition, Faculty of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, Tehran, IRAN (L.V.-M.) Key words: rheumatoid arthritis, probiotics, Lactobacillus casei 01, oxidative stress, clinical trial Objective: Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that causes great pain and disability and increasing oxidative stress in patients. The objective of the present study was to evaluate the effects of probiotics—live microorganisms with many health benefits, including antioxidant properties—on oxidative stress indices of patients with RA. This study is a secondary analysis from a previously published study Methods: In a randomized double-blind placebo-controlled clinical trial, 46 patients with RA were assigned to one of two groups; patients in the probiotic group received a daily capsule containing 108 colony forming units (CFUs) of Lactobacillus casei 01 (L. casei 01), while those in the placebo group took identical capsules containing maltodextrin, for 8 weeks. In the baseline and at the end of the study, anxiety, physical activity levels, and dietary intakes were assessed. Anthropometric parameters, serum malondialdehyde (MDA), total antioxidant capacity (TAC), erythrocyte superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities were measured. Results: There was no significant difference between the two groups for demographic characteristics, anthropometric parameters, physical activity, anxiety levels, or dietary intakes, throughout the course of the study. No significant within- and between-group differences were observed for MDA, TAC, or CAT. SOD activity decreased only in the probiotic group and GPx activity decreased in both study groups (p < 0.05); however, no significant between-group difference was found for these enzymes activities at the end of the study (p > 0.05). Conclusion: No significant effect of L. casei 01 supplementation was observed on the oxidative status of patients with RA, compared to placebo.
INTRODUCTION
and disability in patients. RA prevalence is 0.5–1.0% of adults worldwide [1,2]. Despite intensive work, and though some factors contributing significantly to initiation and propagation of RA have been proposed, its exact etiology is still unknown [1,3].
Rheumatoid arthritis (RA) is an autoimmune inflammatory disease which primarily targets synovial tissues, causing great pain
Address correspondence to: Beitullah Alipour, Associate Professor, Faculty of Nutrition, Tabriz University of Medical Sciences, Attar Neyshapouri Ave., Golgasht St., Tabriz, IRAN. E-mail: [email protected] Abbreviations: 4-NQO D 4-nitroquinoline 1-oxide, ANCOVA D analysis of covariance, BMI D body mass index, CAT D catalase, CBC D complete blood count, CFU D colony forming unit, COX-2 D Cyclooxygenase-2, EDTA D ethylene diamine tetraacetic acid, GCL D glutamate-cysteine ligase, GPx D glutathione peroxidase, GR D glutathione reductase, INT D 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride, IPAQ D international physical activity questionnaire, L. casei D Lactobacillus casei, LDL D Low density lipoprotein, MDA D malondialdehyde, NAFLD D non-alcoholic fatty liver disease, NF-?B D nuclear factor kappa-B, RA D rheumatoid arthritis, ROS D reactive oxygen species, SOD D superoxide dismutase, STAI-Y D Spielberger state-trait anxiety inventory form Y, TAC D total antioxidant capacity, TBARS D Thiobarbituric acid reactive substances, XOD D Xanthine oxidase. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/uacn.
Journal of the American College of Nutrition, Vol. 0, No. 0, 1–9 (2015) Ó American College of Nutrition/ Published by Taylor & Francis Group, LLC 1
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Probiotic Effects on Oxidative Status in RA Oxidative stress has been suggested to play a role in the physiopathology of RA, and a great body of evidence supports the increased level of oxidative stress biomarkers and reduced blood antioxidants in patients with RA [4–11]. Moreover, oxidative stress biomarkers like malondialdehyde (MDA) have been reported to have positive correlation with inflammatory cytokines in subjects with RA [8,9,12–14]. It is suggested that reactive oxygen species (ROS) can lead to inflammatory responses in RA by activating nuclear factor kappa-B (NF-kB) [4]. Therefore, treating patients with antioxidants supplementation may help alleviate symptoms and improve quality of life. Probiotics are live microorganisms which can present the consumer with many health benefits if taken in adequate amounts [15]. Some widely recognized health effects of probiotics include alleviation of gastrointestinal disorders, hypocholesterolemic properties, reduction of blood pressure and regulation of immune system function [16]. Some strains of probiotics also exert antioxidant activities [17,18]; based on results from in vitro and animal studies, some Lactobacillus casei (L. casei) strains can convey such effects. L. casei SY13 had good radical scavenging capacity and inhibited lipid peroxidation in an in vitro study [19]. In another in vitro study investigating cytotoxity of L. casei 01 and its fractions, all the fractions and exopolysaccharides from this strain decreased the cytotoxicity of 4nitroquinoline 1-oxide (4-NQO) against intestine 407 cells; 4-NQO is a carcinogenic substance that may exert its effect through production of reactive oxygen species [20,21]. Rats fed lyophilized L. casei or milk fermented with L. casei had lower levels of thiobarbituric acid reactive substances (TBARS) in their low-density lipoprotein (LDL) [22]. L. casei LAFTI L26 intake resulted in reduced colonic cyclooxygenase-2 (COX-2) expression in rats with induced colitis [23]. Receiving a probiotic product containing L. casei W56 reduced lipid peroxidation in rats with induced acute pancreatitis. The treatment prevented glutathione depletion in the rats as well [24]. Also, this species, in combination with other probiotics, has been successfully used in reducing MDA in patients with nonalcoholic fatty liver disease (NAFLD) [25]. To the best of our knowledge, no clinical trials have been conducted thus far to investigate the effects of probiotic supplementation on oxidative stress biomarkers in patients with RA. Thus, in the present study, we aimed to evaluate the effects of L. casei (a species with probable antioxidant properties) supplementation on MDA, the activities of erythrocyte antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)), and total antioxidant capacity (TAC) in women with RA. This study is a secondary analysis from a previously published study [26].
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MATERIALS AND METHODS Subjects and Study Design The present study was a randomized double-blind placebocontrolled clinical trial. Details of the study protocol, subject recruitment, and exclusion criteria for the patients were previously described [26]. In summary, 60 women with RA established for more than 1 year who had an inactive to moderate level of the disease and were between 20 and 80 years of age, had body mass index (BMI) lower than 40, and followed stable medication for at least the prior 3 months entered the study, if interested. Pregnant or lactating women and those under hormone therapy, suffering from metabolic or gastrointestinal diseases, taking antioxidant, vitamin, fiber, or omega-3 supplements 3 weeks prior to the interventions, smoking or being exposed to cigarette smoke, using antibiotics or other probiotic products, or following a weight reduction diet were excluded from the study. Sample size for the present clinical trial was calculated based on the mean (SD) results for MDA in the study by Kullisaar et al. [27], with a confidence level of 95% and power of 80%; 10 subjects had to be recruited in each intervention group. Thus, the sample size of 22, which had been calculated for the previous study, which assessed the effects of probiotic supplementation on inflammatory biomarkers in women with RA [26], was sufficient for the purpose of the present work. The study was conducted between September and November 2012. The study protocol was fully presented previously [26]. In brief, 30 patients were allocated into each group using a computer-generated blocked randomization list stratified by two factors: menopausal status (premenopausal or postmenopausal) and BMI (30 Kg/m2). At baseline, a demographic questionnaire, the international physical activity questionnaire (IPAQ), the Spielberger statetrait anxiety inventory form Y (STAI-Y), a 24-hour dietary recall questionnaire, and 3 food record questionnaires were completed for the participants. The level of physical activity was assessed by the short form of the IPAQ, considering the activities performed during the previous week, and was categorized as high, moderate, or low [28]. The Spielberger STAI-Y questionnaire consisted of 20 questions regarding the state of anxiety (how the patient felt at the time of filling in the questionnaire) and 20 questions regarding the patient’s anxiety traits (how the patient felt habitually); the scores obtained were converted into categories of anxiety level [29,30]. Usual dietary intakes of the patients were estimated using the mean scores of the calories and nutrients calculated from the four dietary questionnaires. Anthropometric measurements were done, and 8 ml of fasting blood sample was drawn. After 8 weeks of supplementation, the same assessments were performed.
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Probiotic Effects on Oxidative Status in RA Capsules containing a minimum of 108 colony forming units (CFUs) of L. casei 01 (Chr. Hansen, Horsholm, Denmark) and maltodextrin (Shandong, Shandong, China) were the supplements administered to the patients in the probiotic group. The capsules were cultured three times; at baseline, in the middle, and at the end of the intervention course. Bacterial enumeration of the capsules confirmed that the capsules had a minimum of 108 CFUs of L. casei 01 through the whole study course. The participants in the placebo group received identical capsules which contained only the excipient, maltodextrin. Compliance with the study protocol was assessed by capsule count at the end of the study [31].
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Measurement of Oxidative Stress Indices The blood samples were divided into two parts when drawn. Three milliliters were poured into a tube containing the anticoagulant ethylene diamine tetraacetic acid (EDTA), to be used for measurement of complete blood count (CBC) and erythrocyte SOD, GPx and CAT activities. Five milliliters were put into a glass tube from which the serum samples were separated by centrifugation at 3500 rpm for 10 min (Orum Tadjhiz Centrifuge, Iran), at room temperature; in serum samples, MDA and TAC concentrations were measured. Whole blood and serum samples were stored at ¡70 C until analyzed. Erythrocyte SOD activity was measured spectrophotometrically using a Ransod kit (Randox Laboratories, Crumlin, UK). In this method, xanthine and xanthine oxidase (XOD) are used to generate superoxide radicals which react with 2-(4-iodophenyl)-3(4-nitrophenol)-5-phenyltetrazolium chloride (I.N.T.) to form a red formazan color. By the degree of inhibition of this reaction, the SOD activity is measured (Ransod, Superoxide dismutase, Manual). Erythrocyte GPx activity was measured using the spectrophotometric technique and a Ransel kit (Randox Laboratories, Crumlin, UK). In this method, GPx catalyses the oxidation of glutathione by cumene hydroperoxide. The oxidized glutathione is promptly converted to the reduced form in the presence of glutathione reductase and NADPH. This results in oxidation of NADPH to NADPC. The decrease in absorbance at 340 nm is measured (Ransel, Glutathione peroxidase, Manual). Erythrocyte CAT activity was measured spectrophotometrically by the method of Aebi in which H2O2 degradation (2 H2O2 !2 H2O C O2) rate is measured at 240 nm [32]. TAC was determined using a Randox kit (Randox Laboratories, Crumlin, UK) and MDA concentration was determined using the thiobarbituric acid method.
Statistical Analysis Statistical analyses were performed per protocol, by SPSS version 20.0 software (SPSS Inc., Chicago, IL, USA). The results were expressed as mean (SD) for normally distributed quantitative data, median (percentiles 25 and 75) for
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quantitative data not normally distributed, and frequency (percent) for qualitative data. Normality of distribution was tested by the Shapiro-Wilk test. At baseline, the independent samples t test, Mann-Whitney U test, x2, and Fisher’s exact test were used to compare the two groups. The Mann-Whitney U test was used to compare state and trait anxiety categories as well as physical activity level between the two groups both at baseline and at the end of the study; the sign test was used to compare the changes of these variables within groups, throughout the study. For normally distributed data, the independent samples t test and paired samples t test were used to assess between-group differences at baseline and within-group changes throughout the study, respectively; the Mann-Whitney U test and Wilcoxon signed-rank test were used for data not normally distributed. Analysis of covariance (ANCOVA) was used to compare the two groups at the end of the study, adjusting for the baseline measures and menopausal status. Results with p < 0.05 were considered statistically significant.
Ethics All procedures followed in the present clinical trial were according to the guidelines of the ethics committee of Tabriz University of Medical Sciences (no. 9150) and the Helsinki Declaration. Written informed consent was signed by all study participants and the study was registered in the Iranian Registry of Clinical Trials (IRCT), available at: http://www.irct.ir (ID: IRCT201207024105N10).
RESULTS Baseline Characteristics The flow chart of the study is presented in Fig. 1. As previously reported [26], there were no between-group differences for baseline characteristics of the participants (Table 1). Also, no between- or within-group differences were revealed for anthropometric measures. State-trait anxiety and physical activity levels remained almost constant during the study course as well [26].
Dietary Intake No significant differences were found between the two groups for energy and macronutrient intake at baseline, and the within-group changes were insignificant as well, during the intervention period [26]. Since changes in micronutrient intakes with potential oxidant and antioxidant properties could have confounded the results of the study, their intakes were also analyzed. There was no significant between- or withingroup difference for the micronutrient intakes in the study subjects (Table 2).
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Probiotic Effects on Oxidative Status in RA
Fig. 1. Flow chart of the study.
Oxidative Stress Indices Table 3 presents the results for serum MDA and TAC concentrations as well as the erythrocyte SOD, GPx, and CAT activities during the study. Regarding serum MDA and TAC
levels and CAT activity, no within-group changes were observed in either study group, and the between-group differences were also insignificant by the end of the study. The mean percent of changes in MDA concentrations was 23.46% for the
Table 1. Baseline Characteristics of the Ppatients
Age (yrs)y Height (cm)y Weight (Kg)y BMI (Kg/m2)y Duration of RA (yrs)z Menopausal statusz Premenopausal Postmenopausal Educationz Under diploma Diploma Upper diploma Current medication Methotrexatex Hydroxychloroquine{ Prednisolone{
Placebo Group (n D 24)
Probiotic Group (n D 22)
44.29 (9.77) 156.02 (6.40) 68.56 (11.96) 28.08 (4.03) 4.75 (3.0, 9.0)
41.14 (12.65) 158.16 (6.78) 69.29 (11.47) 27.70 (4.16) 5.25 (3.75, 10.0)
17 (70.8) 7 (29.2)
15 (68.2) 7 (31.8)
19 (79.2) 4 (16.7) 1 (4.2)
16 (72.7) 3 (13.6) 3 (13.6)
0.526
20 (83.3) 18 (75.0) 23 (95.8)
15 (68.2) 18 (81.8) 21 (95.5)
0.229 0.725 1.000
P 0.347 0.833 0.753 0.566 0.845
BMI: Body mass index; RA: Rheumatoid arthritis Mean (SD) and median (percentiles 25 and 75) are presented for age and duration of RA, respectively. Frequency (percent) is reported for menopausal status, education, and current medication. y Independent t test. z Mann-Whitney U test. x 2 x test. { Fisher’s exact test.
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Table 2. Micronutrient Intakes in the Two Experimental Groups at Baseline and Throughout the Study
Vit A (RE) Baseline End of study p Vit E (mg) Baseline End of study p Vit C (mg) Baseline End of study p Vit B6 (mg) Baseline End of study p Vit B9 (mg) Baseline End of study p Iron (mg) Baseline End of study p Zinc (mg) Baseline End of study pz Copper (mg) Baseline End of study p Selenium (mg) Baseline End of study p Calcium (mg) Baseline End of study p Phosphorus (mg) Baseline End of study pz
Placebo (n D 24)
Probiotic (n D 22)
P
390.70 (189.95, 667.82) 296.05 (215.55, 654.82) 0.710
429.60 (155.55, 672.72) 403.05 (195.57, 678.75) 0.987
0.921x 0.232*
1.80 (0.77, 3.11) 2.29 (1.34, 3.59) 0.236
1.72 (0.93, 2.54) 2.25 (1.47, 3.92) 0.230
0.904x 0.952*
55.73 (35.30, 106.70) 54.07 (31.43, 92.37) 0.932
52.89 (25.56, 108.77) 49.98 (28.08, 96.81) 0.638
0.895x 0.805*
1.11 (0.70, 1.81) 1.00 (0.77, 1.71) 0.068
0.82 (0.61, 1.36) 0.75 (0.57, 0.96) 0.289
0.173x 0.092*
112.50 (81.30, 159.17) 131.40 (99.84, 185.60) 0.097
96.12 (70.45, 168.50) 112.50 (80.24, 135.50) 0.485
0.629x 0.102*
12.06 (3.33) 12.44 (9.68, 17.18) 0.209
11.97 (9.65, 13.52) 11.74 (3.23) 0.961
0.895x 0.275*
4.95 (2.45) 4.88 (2.02) 0.898
4.69 (2.35) 4.83 (1.74) 0.747
0.712y 0.961*
0.77 (0.46, 0.98) 0.69 (0.51, 0.95) 0.420
0.62 (0.45, 0.87) 0.67 (0.28) 0.626
0.385x 0.405*
0.05 (0.03, 0.11) 0.07 (0.04, 0.09) 0.531
0.07 (0.05, 0.10) 0.07 (0.03, 0.12) 0.917
0.332x 0.648*
331.50 (268.25, 527.30) 335.70 (266.07, 559.20) 0.775
312.00 (181.20, 487.45) 378.90 (243.40, 522.72) 0.390
0.312x 0.712*
504.47 (235.77) 472.04 (172.46) 0.528
504.83 (230.58) 537.66 (261.16) 0.607
0.996y 0.303*
Mean (SD) and Median (percentiles 25 and 75) are presented for normally and not normally distributed measures, respectively. Median of differences (percentiles 25 and 75) are presented for measures not normally distributed, instead of mean difference (95% CI). y Independent t test. z Paired t test. x Mann-Whitney U test. Wilcoxon test. * Based on ANCOVA adjusted for baseline measures.
probiotic group and ¡10.96% for the placebo group (p D 0.904). A mean of 3.59% increase was found for TAC in the probiotic group, which was slightly but insignificantly higher than that in the placebo group, 3.13% (p D 0.886). CAT activity increased by 7.65% and 4.72% in the probiotic and placebo groups, respectively (p D 0.809). SOD activity decreased
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significantly only in the probiotic group and GPx activity was reduced in both groups through the intervention period; however, the between-group differences remained insignificant at the end of the study, for both enzymes. Mean percent of changes for SOD and GPx activities were ¡5.54% and ¡3.30% in the probiotic group, respectively; in the placebo
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Table 3. Effect of Eight Weeks of Probiotic Supplementation as Compared with Placebo on Oxidative Stress Indices in Female Patients with RA
MDA (nmol/ml) Baseline End of study P SOD (U/g Hb) Baseline End of study P GPx (U/g Hb) Baseline End of study P CAT (K/g Hb) Baseline End of study P TAC (mmol/L) Baseline End of study P
Placebo (n D24)
Probiotic (n D 22)
Mean Difference (95% CI)
P
2.57 (1.22) 2.20 (1.17, 2.50) 0.088x
1.98 (1.05) 1.7 (1.67, 1.72) 0.212x
¡0.59 (¡1.27, 0.09) ¡0.18 (¡0.65, 0.29)
0.088y 0.445z
1127.97 (180.37) 1111.16 (206.44) 0.547{
1156.26 (206.95) 1090.35 (198.92) 0.003{
28.29 (¡86.83, 143.40) ¡44.46 (¡113.42, 24.49)
0.623y 0.200z
38.22 (36.07, 40.15) 37.25 (35.11, 39.12) 0.032x
38.53 (3.31) 37.23 (3.24) 0.001{
¡0.33 (¡2.31, 3.00) ¡0.55 (¡2.09, 0.99)
0.652 0.477z
134.32 (99.18, 182.67) 126.77 (86.37, 166.22) 0.116x
122.55 (96.02, 165.65) 113.55 (75.10, 150.88) 0.189x
¡10.12 (¡45.64, 73.92) ¡6.69 (¡50.95, 37.56)
0.725 0.762z
1.13 (0.20) 1.15 (0.16) 0.350{
1.19 (0.19) 1.21 (0.14) 0.401{
0.06 (¡0.06, 0.17) 0.03 (¡0.03, 0.09)
0.334y 0.359z
MDA: Malondialdehyde; SOD: Superoxide dismutase; GPx: Glutathione peroxidase; CAT: Catalase; TAC: Total antioxidant capacity. Mean (SD) and median (percentiles 25 and 75) are presented for normally and not normally distributed measures, respectively. Median of differences (percentiles 25 and 75) are presented instead of mean difference (95% CI), when Mann-Whitney U test applied. p-values of statistical significance (p < 0.05) are presented in bold. y Independent t test. z Based on ANCOVA adjusted for baseline measures and menopausal status. x Wilcoxon test. { Paired t test. Mann-Whitney U test.
group, SOD activity decreased by 1.22% and GPx activity diminished by 1.85%. No significant differences in percent of changes were seen between the two groups for SOD and GPx activities (p D 0.169 and p D 0.613, respectively).
DISCUSSION Although we previously reported promising effects of L. casei 01 on disease activity and inflammatory status of women with RA [26], we failed to observe beneficial effects of this supplementation on the oxidative status of patients. It has frequently been reported that the gut microbiota is altered in patients with RA [33–36] and the contributory effect of such alteration in initiation of the disease has been revealed by animal studies [37]. On the other hand, oxidative stress has been suggested to be implicated in the disease pathogenesis [4] and is reported to play a role in gut microbiota changes as well [38]. Thus, we hypothesized that supplementing the patients with a probiotic strain with probable antioxidant effects [20] may both positively affect the gut flora and reduce oxidative stress in the subjects. Drinking 150 g/day of goat milk fermented with L. fermentum for 21 days lowered glutathione redox ratio and
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enhanced total antioxidative activity in healthy subjects. The authors suggested that the high level of reduced glutathione content of the administered probiotic was principally responsible for the observed effects; other thiol compounds were assumed to play a part as well [27]. Supplementation of patients with NAFLD and alcoholic liver cirrhosis (AC) with the probiotic preparation VSL#3 significantly improved MDA and 4-hydroxynonenal (4-HNE) [25]. Chamari et al. showed that, in healthy women, 6 weeks of probiotic yogurt consumption significantly increased CAT activity, in comparison with conventional yogurt; SOD activity increased in both study groups [39]. In athletes with increased level of oxidative stress resulting from intensive exercises, taking a probiotic mixture containing L. rhamnosus IMC 501 and L. paracasei IMC 502 for 4 weeks significantly reduced reactive oxygen metabolites (ROM) and increased biological antioxidant potential (BAP); both intact cells and intracellular cell free extracts inhibited lipid peroxidation [40]. Nine weeks of using probiotic yogurt increased plasma glutathione and erythrocyte glutathione reductase (GR), but did not significantly affect other oxidative stress indices in pregnant women. The positive results obtained in this study were attributed to up-regulation of antioxidant enzymes and reduction of inflammatory cytokines (which produce oxygen radicals and increase oxidative stress) [41].
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Probiotic Effects on Oxidative Status in RA The probable mechanisms through which probiotics can exert their antioxidative effects include metal ion chelation, inhibition of ascorbate autoxidation, reduction and scavenging of free radicals, lowering levels of oxidized LDL, as well as reducing glutathione redox ratio in blood, gut, and skin. Moreover, probiotics have been found to increase glutathione levels through either releasing it from other tissues or increasing its synthesis by augmented glutamate-cysteine ligase (GCL) activity and increased mRNA expression of both of the GCL subunits [17, 23–24, 41–46]. Despite the previous studies which reported beneficial effects of probiotics in reducing oxidative stress, we failed to obtain such results in our clinical trial. Our results were in accordance with those of Fabian and Elmadfa who administered 100 g of probiotic yogurt containing 3.6£108 cfu/g of L. casei DN-114 001 and found no significant effects of the treatment in healthy women [47]. The different strain administered in our clinical trial is most likely to be responsible for the disagreement between our results and the others. In contrast to the studies that reported antioxidant effects for some strains of L. casei, and the study that found potential antioxidant properties for L. casei 01 [19,20, 22–24, 48], the subspecies we applied in our investigation (L. casei 01) seems not to possess the very characteristic, either at all or not to an extent that would be revealed at the low dosage (108 CFU/day) and short treatment duration (8 weeks) of our study. The probiotic dosage of our study supplements was lower than that of most of the previous clinical trials. It is probable that higher dosages of L. casei 01 are required to confer the antioxidant effects in consumers; this needs to be investigated in future studies. As a secondary analysis, our study duration was limited to 8 weeks. The enzyme content of mature erythrocytes remains constant during their life span, which is about 120 days [49]. Therefore, it may be estimated that during the 56 days of our treatment, only half of the erythrocytes in blood stream are those in which enzyme content and activity level have been affected by the supplementation. Given that, it is less likely that our probiotic strain (L. casei 01), with apparently lower antioxidant potential compared to the other strains of L. casei species, could exert its full effects. Li et al. showed in an in vitro study that L. casei 01 could convert procyanidins to more effective antioxidant products [50]; thus, it is probable that consuming probiotic foods that are also rich in antioxidants is more effective than taking probiotic supplements in reducing oxidative stress indices. L. casei 01 has been shown to sufficiently resist gastric and intestinal pH and properly adhere to IEC-6 epithelial cells (which is an in vitro indicator of proper colonization in the gut) [51–53]. A major limitation to the present study was that the recovery of the probiotic bacteria was not assessed in feces, because most patients were unwilling to provide fecal samples. However, since the confounding factors were controlled (through keeping physical activity, anxiety, and nutritional intake constant
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throughout the study) and the ANCOVA analyses were used to compare the groups at the end of the study, it is most probable that the within-group changes and between-group differences are attributable to the treatment. On the other hand, the primary analyses for the present clinical trial [26] had shown positive effects of the treatment in patients; therefore, it is less likely that insignificant results in the present work are due to inefficient colonization of the administered probiotic bacteria. Along with the limitations of our study mentioned above, our work had a number of strength points; we considered the intakes of antioxidant nutrients to make sure that changes in dietary intake did not confound our results. Furthermore, evaluation of changes in physical activity and state-trait anxiety levels throughout the study course allowed for elimination of the possibility that alterations in these parameters had affected the oxidative status of the participants.
CONCLUSION We observed no significant effects of an 8week course of L. casei 01 supplementation on oxidative stress indices in women with RA. It is suggested that longer treatment durations be considered in future studies. Investigating the effects of other probiotic strains with confirmed antioxidant effects and at various dosages is encouraged.
ACKNOWLEDGMENTS We would like to thank the Management of Nutrition Faculty and the Drug Applied Research Center of Tabriz University of Medical Sciences, Tabriz, Iran, for supporting the laboratory tests. Special gratitude should go to the subjects who participated in the study, to Dr. Mohammad AsghariJafarabadi, who gave valuable advice on statistical analyses, and to Dr. Davoud Hassanzadeh, for helping us with preparation of the study supplements. The authors declare herein that they have no conflicts of interest.
FUNDING The present study was funded by the Research Vice Chancellor of Tabriz University of Medical Sciences, Tabriz, Iran.
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45. Lutgendorff F, Nijmeijer RM, Sandstrom PA, Trulsson LM, Magnusson KE, Timmerman HM, van Minnen LP, Rijkers GT, Gooszen HG, Akkermans LMA, S€oderholm JD: Probiotics prevent intestinal barrier dysfunction in acute pancreatitis in rats via induction of ileal mucosal glutathione biosynthesis. PLoS One 4: e4512, 2009. 46. Musenga A, Mandrioli R, Bonifazi P, Kenndler E, Pompei A, Raggi MA: Sensitive and selective determination of glutathione in probiotic bacteria by capillary electrophoresis-laser induced fluorescence. Anal Bioanal Chem 387:917–924, 2007. 47. Fabian E, Elmadfa I: The effect of daily consumption of probiotic and conventional yoghurt on oxidant and antioxidant parameters in plasma of young healthy women. Int J Vitam Nutr Res 77 (2):79–88, 2007. 48. Amdekar S, Singh V, Kumar A, Sharma P, Singh R: Lactobacillus casei and Lactobacillus acidophilus regulate inflammatory pathway and improve antioxidant status in collagen-induced arthritic rats. J Interferon Cytokine Res 33(1):1–8, 2013. 49. Pandey KB, Rizvi SI: Markers of oxidative stress in erythrocytes and plasma during aging in humans. Oxid Med Cell Longev 3 (1):2–12, 2010. 50. Li S, Chen L, Yang T, Wu Q, Lv Z, Xie B, Sun Z: Increasing antioxidant activity of procyanidin extracts from the pericarp of Litchi chinensis processing waste by two Probiotic Bacteria Bioconversions. J Agric Food Chem 61:2506–2512, 2013. 51. Homayouni-Rad A: Selection of appropriate probiotic strains in production of functional ice-cream. Microb Biotechnolog J Islamic Azad Univ 3(8):49–56, 2011 [in Persian]. 52. Chan ES, Lee PP, Ravindra P, Krishnaiah K, Voo WP: A standard quantitative method to measure acid tolerance of probiotic cells. App Microbiol Biotechnol 86(1):385–391, 2010. 53. Both E, Gyorgy E, Kibedi-Szabo CZ, Tamas E, Abraham B, Miklossy I, Lanyi S: Acid and bile tolerance, adhesion to epithelial cells of probiotic microorganisms. U.P.B. Sci Bull Series B 72 (2):37–44, 2010.
Received May 23, 2014; accepted August 25, 2014.
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Journal of the American College of Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uacn20
Effects of Probiotic Supplementation on Oxidative Stress Indices in Women with Rheumatoid Arthritis: A Randomized Double-Blind Clinical Trial a
b
c
Elnaz Vaghef-Mehrabany MS , Aziz Homayouni-Rad PhD , Beitullah Alipour PhD , Sakinehd
f
e
Khatoun Sharif PhD , Leila Vaghef-Mehrabany MS & Serour Alipour-Ajiry MS a
Department of Nutrition, Biochemistry and Diet Therapy, Tabriz University of Medical Sciences, Tabriz, IRAN b
Department of Food Science and Technology, Tabriz University of Medical Sciences, Tabriz, IRAN
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c
Department of Community Nutrition, Tabriz University of Medical Sciences, Tabriz, IRAN
d
Rheumatology Department, Tabriz University of Medical Sciences, Tabriz, IRAN
e
Faculty of Nutrition, Research Vice Chancellor, Tabriz University of Medical Sciences, Tabriz, IRAN f
Department of Clinical Nutrition, Faculty of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, Tabriz, IRAN Published online: 09 Apr 2015.
To cite this article: Elnaz Vaghef-Mehrabany MS, Aziz Homayouni-Rad PhD, Beitullah Alipour PhD, Sakineh-Khatoun Sharif PhD, Leila Vaghef-Mehrabany MS & Serour Alipour-Ajiry MS (2015): Effects of Probiotic Supplementation on Oxidative Stress Indices in Women with Rheumatoid Arthritis: A Randomized Double-Blind Clinical Trial, Journal of the American College of Nutrition, DOI: 10.1080/07315724.2014.959208 To link to this article: http://dx.doi.org/10.1080/07315724.2014.959208
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Original Research
Effects of Probiotic Supplementation on Oxidative Stress Indices in Women with Rheumatoid Arthritis: A Randomized Double-Blind Clinical Trial Elnaz Vaghef-Mehrabany, MS, Aziz Homayouni-Rad, PhD, Beitullah Alipour, PhD, Sakineh-Khatoun Sharif, PhD, Leila Vaghef-Mehrabany, MS, and Serour Alipour-Ajiry, MS
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Department of Nutrition, Biochemistry and Diet Therapy (E.V.-M.), Department of Food Science and Technology (A.H.-R.), Department of Community Nutrition (B.A.), Rheumatology Department (S.-K.S.), Faculty of Nutrition, Research Vice Chancellor (S.A.-A.), Tabriz University of Medical Sciences, Tabriz, IRAN; Department of Clinical Nutrition, Faculty of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, Tehran, IRAN (L.V.-M.) Key words: rheumatoid arthritis, probiotics, Lactobacillus casei 01, oxidative stress, clinical trial Objective: Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that causes great pain and disability and increasing oxidative stress in patients. The objective of the present study was to evaluate the effects of probiotics—live microorganisms with many health benefits, including antioxidant properties—on oxidative stress indices of patients with RA. This study is a secondary analysis from a previously published study Methods: In a randomized double-blind placebo-controlled clinical trial, 46 patients with RA were assigned to one of two groups; patients in the probiotic group received a daily capsule containing 108 colony forming units (CFUs) of Lactobacillus casei 01 (L. casei 01), while those in the placebo group took identical capsules containing maltodextrin, for 8 weeks. In the baseline and at the end of the study, anxiety, physical activity levels, and dietary intakes were assessed. Anthropometric parameters, serum malondialdehyde (MDA), total antioxidant capacity (TAC), erythrocyte superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities were measured. Results: There was no significant difference between the two groups for demographic characteristics, anthropometric parameters, physical activity, anxiety levels, or dietary intakes, throughout the course of the study. No significant within- and between-group differences were observed for MDA, TAC, or CAT. SOD activity decreased only in the probiotic group and GPx activity decreased in both study groups (p < 0.05); however, no significant between-group difference was found for these enzymes activities at the end of the study (p > 0.05). Conclusion: No significant effect of L. casei 01 supplementation was observed on the oxidative status of patients with RA, compared to placebo.
INTRODUCTION
and disability in patients. RA prevalence is 0.5–1.0% of adults worldwide [1,2]. Despite intensive work, and though some factors contributing significantly to initiation and propagation of RA have been proposed, its exact etiology is still unknown [1,3].
Rheumatoid arthritis (RA) is an autoimmune inflammatory disease which primarily targets synovial tissues, causing great pain
Address correspondence to: Beitullah Alipour, Associate Professor, Faculty of Nutrition, Tabriz University of Medical Sciences, Attar Neyshapouri Ave., Golgasht St., Tabriz, IRAN. E-mail: [email protected] Abbreviations: 4-NQO D 4-nitroquinoline 1-oxide, ANCOVA D analysis of covariance, BMI D body mass index, CAT D catalase, CBC D complete blood count, CFU D colony forming unit, COX-2 D Cyclooxygenase-2, EDTA D ethylene diamine tetraacetic acid, GCL D glutamate-cysteine ligase, GPx D glutathione peroxidase, GR D glutathione reductase, INT D 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride, IPAQ D international physical activity questionnaire, L. casei D Lactobacillus casei, LDL D Low density lipoprotein, MDA D malondialdehyde, NAFLD D non-alcoholic fatty liver disease, NF-?B D nuclear factor kappa-B, RA D rheumatoid arthritis, ROS D reactive oxygen species, SOD D superoxide dismutase, STAI-Y D Spielberger state-trait anxiety inventory form Y, TAC D total antioxidant capacity, TBARS D Thiobarbituric acid reactive substances, XOD D Xanthine oxidase. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/uacn.
Journal of the American College of Nutrition, Vol. 0, No. 0, 1–9 (2015) Ó American College of Nutrition/ Published by Taylor & Francis Group, LLC 1
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Probiotic Effects on Oxidative Status in RA Oxidative stress has been suggested to play a role in the physiopathology of RA, and a great body of evidence supports the increased level of oxidative stress biomarkers and reduced blood antioxidants in patients with RA [4–11]. Moreover, oxidative stress biomarkers like malondialdehyde (MDA) have been reported to have positive correlation with inflammatory cytokines in subjects with RA [8,9,12–14]. It is suggested that reactive oxygen species (ROS) can lead to inflammatory responses in RA by activating nuclear factor kappa-B (NF-kB) [4]. Therefore, treating patients with antioxidants supplementation may help alleviate symptoms and improve quality of life. Probiotics are live microorganisms which can present the consumer with many health benefits if taken in adequate amounts [15]. Some widely recognized health effects of probiotics include alleviation of gastrointestinal disorders, hypocholesterolemic properties, reduction of blood pressure and regulation of immune system function [16]. Some strains of probiotics also exert antioxidant activities [17,18]; based on results from in vitro and animal studies, some Lactobacillus casei (L. casei) strains can convey such effects. L. casei SY13 had good radical scavenging capacity and inhibited lipid peroxidation in an in vitro study [19]. In another in vitro study investigating cytotoxity of L. casei 01 and its fractions, all the fractions and exopolysaccharides from this strain decreased the cytotoxicity of 4nitroquinoline 1-oxide (4-NQO) against intestine 407 cells; 4-NQO is a carcinogenic substance that may exert its effect through production of reactive oxygen species [20,21]. Rats fed lyophilized L. casei or milk fermented with L. casei had lower levels of thiobarbituric acid reactive substances (TBARS) in their low-density lipoprotein (LDL) [22]. L. casei LAFTI L26 intake resulted in reduced colonic cyclooxygenase-2 (COX-2) expression in rats with induced colitis [23]. Receiving a probiotic product containing L. casei W56 reduced lipid peroxidation in rats with induced acute pancreatitis. The treatment prevented glutathione depletion in the rats as well [24]. Also, this species, in combination with other probiotics, has been successfully used in reducing MDA in patients with nonalcoholic fatty liver disease (NAFLD) [25]. To the best of our knowledge, no clinical trials have been conducted thus far to investigate the effects of probiotic supplementation on oxidative stress biomarkers in patients with RA. Thus, in the present study, we aimed to evaluate the effects of L. casei (a species with probable antioxidant properties) supplementation on MDA, the activities of erythrocyte antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)), and total antioxidant capacity (TAC) in women with RA. This study is a secondary analysis from a previously published study [26].
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MATERIALS AND METHODS Subjects and Study Design The present study was a randomized double-blind placebocontrolled clinical trial. Details of the study protocol, subject recruitment, and exclusion criteria for the patients were previously described [26]. In summary, 60 women with RA established for more than 1 year who had an inactive to moderate level of the disease and were between 20 and 80 years of age, had body mass index (BMI) lower than 40, and followed stable medication for at least the prior 3 months entered the study, if interested. Pregnant or lactating women and those under hormone therapy, suffering from metabolic or gastrointestinal diseases, taking antioxidant, vitamin, fiber, or omega-3 supplements 3 weeks prior to the interventions, smoking or being exposed to cigarette smoke, using antibiotics or other probiotic products, or following a weight reduction diet were excluded from the study. Sample size for the present clinical trial was calculated based on the mean (SD) results for MDA in the study by Kullisaar et al. [27], with a confidence level of 95% and power of 80%; 10 subjects had to be recruited in each intervention group. Thus, the sample size of 22, which had been calculated for the previous study, which assessed the effects of probiotic supplementation on inflammatory biomarkers in women with RA [26], was sufficient for the purpose of the present work. The study was conducted between September and November 2012. The study protocol was fully presented previously [26]. In brief, 30 patients were allocated into each group using a computer-generated blocked randomization list stratified by two factors: menopausal status (premenopausal or postmenopausal) and BMI (30 Kg/m2). At baseline, a demographic questionnaire, the international physical activity questionnaire (IPAQ), the Spielberger statetrait anxiety inventory form Y (STAI-Y), a 24-hour dietary recall questionnaire, and 3 food record questionnaires were completed for the participants. The level of physical activity was assessed by the short form of the IPAQ, considering the activities performed during the previous week, and was categorized as high, moderate, or low [28]. The Spielberger STAI-Y questionnaire consisted of 20 questions regarding the state of anxiety (how the patient felt at the time of filling in the questionnaire) and 20 questions regarding the patient’s anxiety traits (how the patient felt habitually); the scores obtained were converted into categories of anxiety level [29,30]. Usual dietary intakes of the patients were estimated using the mean scores of the calories and nutrients calculated from the four dietary questionnaires. Anthropometric measurements were done, and 8 ml of fasting blood sample was drawn. After 8 weeks of supplementation, the same assessments were performed.
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Probiotic Effects on Oxidative Status in RA Capsules containing a minimum of 108 colony forming units (CFUs) of L. casei 01 (Chr. Hansen, Horsholm, Denmark) and maltodextrin (Shandong, Shandong, China) were the supplements administered to the patients in the probiotic group. The capsules were cultured three times; at baseline, in the middle, and at the end of the intervention course. Bacterial enumeration of the capsules confirmed that the capsules had a minimum of 108 CFUs of L. casei 01 through the whole study course. The participants in the placebo group received identical capsules which contained only the excipient, maltodextrin. Compliance with the study protocol was assessed by capsule count at the end of the study [31].
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Measurement of Oxidative Stress Indices The blood samples were divided into two parts when drawn. Three milliliters were poured into a tube containing the anticoagulant ethylene diamine tetraacetic acid (EDTA), to be used for measurement of complete blood count (CBC) and erythrocyte SOD, GPx and CAT activities. Five milliliters were put into a glass tube from which the serum samples were separated by centrifugation at 3500 rpm for 10 min (Orum Tadjhiz Centrifuge, Iran), at room temperature; in serum samples, MDA and TAC concentrations were measured. Whole blood and serum samples were stored at ¡70 C until analyzed. Erythrocyte SOD activity was measured spectrophotometrically using a Ransod kit (Randox Laboratories, Crumlin, UK). In this method, xanthine and xanthine oxidase (XOD) are used to generate superoxide radicals which react with 2-(4-iodophenyl)-3(4-nitrophenol)-5-phenyltetrazolium chloride (I.N.T.) to form a red formazan color. By the degree of inhibition of this reaction, the SOD activity is measured (Ransod, Superoxide dismutase, Manual). Erythrocyte GPx activity was measured using the spectrophotometric technique and a Ransel kit (Randox Laboratories, Crumlin, UK). In this method, GPx catalyses the oxidation of glutathione by cumene hydroperoxide. The oxidized glutathione is promptly converted to the reduced form in the presence of glutathione reductase and NADPH. This results in oxidation of NADPH to NADPC. The decrease in absorbance at 340 nm is measured (Ransel, Glutathione peroxidase, Manual). Erythrocyte CAT activity was measured spectrophotometrically by the method of Aebi in which H2O2 degradation (2 H2O2 !2 H2O C O2) rate is measured at 240 nm [32]. TAC was determined using a Randox kit (Randox Laboratories, Crumlin, UK) and MDA concentration was determined using the thiobarbituric acid method.
Statistical Analysis Statistical analyses were performed per protocol, by SPSS version 20.0 software (SPSS Inc., Chicago, IL, USA). The results were expressed as mean (SD) for normally distributed quantitative data, median (percentiles 25 and 75) for
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quantitative data not normally distributed, and frequency (percent) for qualitative data. Normality of distribution was tested by the Shapiro-Wilk test. At baseline, the independent samples t test, Mann-Whitney U test, x2, and Fisher’s exact test were used to compare the two groups. The Mann-Whitney U test was used to compare state and trait anxiety categories as well as physical activity level between the two groups both at baseline and at the end of the study; the sign test was used to compare the changes of these variables within groups, throughout the study. For normally distributed data, the independent samples t test and paired samples t test were used to assess between-group differences at baseline and within-group changes throughout the study, respectively; the Mann-Whitney U test and Wilcoxon signed-rank test were used for data not normally distributed. Analysis of covariance (ANCOVA) was used to compare the two groups at the end of the study, adjusting for the baseline measures and menopausal status. Results with p < 0.05 were considered statistically significant.
Ethics All procedures followed in the present clinical trial were according to the guidelines of the ethics committee of Tabriz University of Medical Sciences (no. 9150) and the Helsinki Declaration. Written informed consent was signed by all study participants and the study was registered in the Iranian Registry of Clinical Trials (IRCT), available at: http://www.irct.ir (ID: IRCT201207024105N10).
RESULTS Baseline Characteristics The flow chart of the study is presented in Fig. 1. As previously reported [26], there were no between-group differences for baseline characteristics of the participants (Table 1). Also, no between- or within-group differences were revealed for anthropometric measures. State-trait anxiety and physical activity levels remained almost constant during the study course as well [26].
Dietary Intake No significant differences were found between the two groups for energy and macronutrient intake at baseline, and the within-group changes were insignificant as well, during the intervention period [26]. Since changes in micronutrient intakes with potential oxidant and antioxidant properties could have confounded the results of the study, their intakes were also analyzed. There was no significant between- or withingroup difference for the micronutrient intakes in the study subjects (Table 2).
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Probiotic Effects on Oxidative Status in RA
Fig. 1. Flow chart of the study.
Oxidative Stress Indices Table 3 presents the results for serum MDA and TAC concentrations as well as the erythrocyte SOD, GPx, and CAT activities during the study. Regarding serum MDA and TAC
levels and CAT activity, no within-group changes were observed in either study group, and the between-group differences were also insignificant by the end of the study. The mean percent of changes in MDA concentrations was 23.46% for the
Table 1. Baseline Characteristics of the Ppatients
Age (yrs)y Height (cm)y Weight (Kg)y BMI (Kg/m2)y Duration of RA (yrs)z Menopausal statusz Premenopausal Postmenopausal Educationz Under diploma Diploma Upper diploma Current medication Methotrexatex Hydroxychloroquine{ Prednisolone{
Placebo Group (n D 24)
Probiotic Group (n D 22)
44.29 (9.77) 156.02 (6.40) 68.56 (11.96) 28.08 (4.03) 4.75 (3.0, 9.0)
41.14 (12.65) 158.16 (6.78) 69.29 (11.47) 27.70 (4.16) 5.25 (3.75, 10.0)
17 (70.8) 7 (29.2)
15 (68.2) 7 (31.8)
19 (79.2) 4 (16.7) 1 (4.2)
16 (72.7) 3 (13.6) 3 (13.6)
0.526
20 (83.3) 18 (75.0) 23 (95.8)
15 (68.2) 18 (81.8) 21 (95.5)
0.229 0.725 1.000
P 0.347 0.833 0.753 0.566 0.845
BMI: Body mass index; RA: Rheumatoid arthritis Mean (SD) and median (percentiles 25 and 75) are presented for age and duration of RA, respectively. Frequency (percent) is reported for menopausal status, education, and current medication. y Independent t test. z Mann-Whitney U test. x 2 x test. { Fisher’s exact test.
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Table 2. Micronutrient Intakes in the Two Experimental Groups at Baseline and Throughout the Study
Vit A (RE) Baseline End of study p Vit E (mg) Baseline End of study p Vit C (mg) Baseline End of study p Vit B6 (mg) Baseline End of study p Vit B9 (mg) Baseline End of study p Iron (mg) Baseline End of study p Zinc (mg) Baseline End of study pz Copper (mg) Baseline End of study p Selenium (mg) Baseline End of study p Calcium (mg) Baseline End of study p Phosphorus (mg) Baseline End of study pz
Placebo (n D 24)
Probiotic (n D 22)
P
390.70 (189.95, 667.82) 296.05 (215.55, 654.82) 0.710
429.60 (155.55, 672.72) 403.05 (195.57, 678.75) 0.987
0.921x 0.232*
1.80 (0.77, 3.11) 2.29 (1.34, 3.59) 0.236
1.72 (0.93, 2.54) 2.25 (1.47, 3.92) 0.230
0.904x 0.952*
55.73 (35.30, 106.70) 54.07 (31.43, 92.37) 0.932
52.89 (25.56, 108.77) 49.98 (28.08, 96.81) 0.638
0.895x 0.805*
1.11 (0.70, 1.81) 1.00 (0.77, 1.71) 0.068
0.82 (0.61, 1.36) 0.75 (0.57, 0.96) 0.289
0.173x 0.092*
112.50 (81.30, 159.17) 131.40 (99.84, 185.60) 0.097
96.12 (70.45, 168.50) 112.50 (80.24, 135.50) 0.485
0.629x 0.102*
12.06 (3.33) 12.44 (9.68, 17.18) 0.209
11.97 (9.65, 13.52) 11.74 (3.23) 0.961
0.895x 0.275*
4.95 (2.45) 4.88 (2.02) 0.898
4.69 (2.35) 4.83 (1.74) 0.747
0.712y 0.961*
0.77 (0.46, 0.98) 0.69 (0.51, 0.95) 0.420
0.62 (0.45, 0.87) 0.67 (0.28) 0.626
0.385x 0.405*
0.05 (0.03, 0.11) 0.07 (0.04, 0.09) 0.531
0.07 (0.05, 0.10) 0.07 (0.03, 0.12) 0.917
0.332x 0.648*
331.50 (268.25, 527.30) 335.70 (266.07, 559.20) 0.775
312.00 (181.20, 487.45) 378.90 (243.40, 522.72) 0.390
0.312x 0.712*
504.47 (235.77) 472.04 (172.46) 0.528
504.83 (230.58) 537.66 (261.16) 0.607
0.996y 0.303*
Mean (SD) and Median (percentiles 25 and 75) are presented for normally and not normally distributed measures, respectively. Median of differences (percentiles 25 and 75) are presented for measures not normally distributed, instead of mean difference (95% CI). y Independent t test. z Paired t test. x Mann-Whitney U test. Wilcoxon test. * Based on ANCOVA adjusted for baseline measures.
probiotic group and ¡10.96% for the placebo group (p D 0.904). A mean of 3.59% increase was found for TAC in the probiotic group, which was slightly but insignificantly higher than that in the placebo group, 3.13% (p D 0.886). CAT activity increased by 7.65% and 4.72% in the probiotic and placebo groups, respectively (p D 0.809). SOD activity decreased
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significantly only in the probiotic group and GPx activity was reduced in both groups through the intervention period; however, the between-group differences remained insignificant at the end of the study, for both enzymes. Mean percent of changes for SOD and GPx activities were ¡5.54% and ¡3.30% in the probiotic group, respectively; in the placebo
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Table 3. Effect of Eight Weeks of Probiotic Supplementation as Compared with Placebo on Oxidative Stress Indices in Female Patients with RA
MDA (nmol/ml) Baseline End of study P SOD (U/g Hb) Baseline End of study P GPx (U/g Hb) Baseline End of study P CAT (K/g Hb) Baseline End of study P TAC (mmol/L) Baseline End of study P
Placebo (n D24)
Probiotic (n D 22)
Mean Difference (95% CI)
P
2.57 (1.22) 2.20 (1.17, 2.50) 0.088x
1.98 (1.05) 1.7 (1.67, 1.72) 0.212x
¡0.59 (¡1.27, 0.09) ¡0.18 (¡0.65, 0.29)
0.088y 0.445z
1127.97 (180.37) 1111.16 (206.44) 0.547{
1156.26 (206.95) 1090.35 (198.92) 0.003{
28.29 (¡86.83, 143.40) ¡44.46 (¡113.42, 24.49)
0.623y 0.200z
38.22 (36.07, 40.15) 37.25 (35.11, 39.12) 0.032x
38.53 (3.31) 37.23 (3.24) 0.001{
¡0.33 (¡2.31, 3.00) ¡0.55 (¡2.09, 0.99)
0.652 0.477z
134.32 (99.18, 182.67) 126.77 (86.37, 166.22) 0.116x
122.55 (96.02, 165.65) 113.55 (75.10, 150.88) 0.189x
¡10.12 (¡45.64, 73.92) ¡6.69 (¡50.95, 37.56)
0.725 0.762z
1.13 (0.20) 1.15 (0.16) 0.350{
1.19 (0.19) 1.21 (0.14) 0.401{
0.06 (¡0.06, 0.17) 0.03 (¡0.03, 0.09)
0.334y 0.359z
MDA: Malondialdehyde; SOD: Superoxide dismutase; GPx: Glutathione peroxidase; CAT: Catalase; TAC: Total antioxidant capacity. Mean (SD) and median (percentiles 25 and 75) are presented for normally and not normally distributed measures, respectively. Median of differences (percentiles 25 and 75) are presented instead of mean difference (95% CI), when Mann-Whitney U test applied. p-values of statistical significance (p < 0.05) are presented in bold. y Independent t test. z Based on ANCOVA adjusted for baseline measures and menopausal status. x Wilcoxon test. { Paired t test. Mann-Whitney U test.
group, SOD activity decreased by 1.22% and GPx activity diminished by 1.85%. No significant differences in percent of changes were seen between the two groups for SOD and GPx activities (p D 0.169 and p D 0.613, respectively).
DISCUSSION Although we previously reported promising effects of L. casei 01 on disease activity and inflammatory status of women with RA [26], we failed to observe beneficial effects of this supplementation on the oxidative status of patients. It has frequently been reported that the gut microbiota is altered in patients with RA [33–36] and the contributory effect of such alteration in initiation of the disease has been revealed by animal studies [37]. On the other hand, oxidative stress has been suggested to be implicated in the disease pathogenesis [4] and is reported to play a role in gut microbiota changes as well [38]. Thus, we hypothesized that supplementing the patients with a probiotic strain with probable antioxidant effects [20] may both positively affect the gut flora and reduce oxidative stress in the subjects. Drinking 150 g/day of goat milk fermented with L. fermentum for 21 days lowered glutathione redox ratio and
6
enhanced total antioxidative activity in healthy subjects. The authors suggested that the high level of reduced glutathione content of the administered probiotic was principally responsible for the observed effects; other thiol compounds were assumed to play a part as well [27]. Supplementation of patients with NAFLD and alcoholic liver cirrhosis (AC) with the probiotic preparation VSL#3 significantly improved MDA and 4-hydroxynonenal (4-HNE) [25]. Chamari et al. showed that, in healthy women, 6 weeks of probiotic yogurt consumption significantly increased CAT activity, in comparison with conventional yogurt; SOD activity increased in both study groups [39]. In athletes with increased level of oxidative stress resulting from intensive exercises, taking a probiotic mixture containing L. rhamnosus IMC 501 and L. paracasei IMC 502 for 4 weeks significantly reduced reactive oxygen metabolites (ROM) and increased biological antioxidant potential (BAP); both intact cells and intracellular cell free extracts inhibited lipid peroxidation [40]. Nine weeks of using probiotic yogurt increased plasma glutathione and erythrocyte glutathione reductase (GR), but did not significantly affect other oxidative stress indices in pregnant women. The positive results obtained in this study were attributed to up-regulation of antioxidant enzymes and reduction of inflammatory cytokines (which produce oxygen radicals and increase oxidative stress) [41].
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Probiotic Effects on Oxidative Status in RA The probable mechanisms through which probiotics can exert their antioxidative effects include metal ion chelation, inhibition of ascorbate autoxidation, reduction and scavenging of free radicals, lowering levels of oxidized LDL, as well as reducing glutathione redox ratio in blood, gut, and skin. Moreover, probiotics have been found to increase glutathione levels through either releasing it from other tissues or increasing its synthesis by augmented glutamate-cysteine ligase (GCL) activity and increased mRNA expression of both of the GCL subunits [17, 23–24, 41–46]. Despite the previous studies which reported beneficial effects of probiotics in reducing oxidative stress, we failed to obtain such results in our clinical trial. Our results were in accordance with those of Fabian and Elmadfa who administered 100 g of probiotic yogurt containing 3.6£108 cfu/g of L. casei DN-114 001 and found no significant effects of the treatment in healthy women [47]. The different strain administered in our clinical trial is most likely to be responsible for the disagreement between our results and the others. In contrast to the studies that reported antioxidant effects for some strains of L. casei, and the study that found potential antioxidant properties for L. casei 01 [19,20, 22–24, 48], the subspecies we applied in our investigation (L. casei 01) seems not to possess the very characteristic, either at all or not to an extent that would be revealed at the low dosage (108 CFU/day) and short treatment duration (8 weeks) of our study. The probiotic dosage of our study supplements was lower than that of most of the previous clinical trials. It is probable that higher dosages of L. casei 01 are required to confer the antioxidant effects in consumers; this needs to be investigated in future studies. As a secondary analysis, our study duration was limited to 8 weeks. The enzyme content of mature erythrocytes remains constant during their life span, which is about 120 days [49]. Therefore, it may be estimated that during the 56 days of our treatment, only half of the erythrocytes in blood stream are those in which enzyme content and activity level have been affected by the supplementation. Given that, it is less likely that our probiotic strain (L. casei 01), with apparently lower antioxidant potential compared to the other strains of L. casei species, could exert its full effects. Li et al. showed in an in vitro study that L. casei 01 could convert procyanidins to more effective antioxidant products [50]; thus, it is probable that consuming probiotic foods that are also rich in antioxidants is more effective than taking probiotic supplements in reducing oxidative stress indices. L. casei 01 has been shown to sufficiently resist gastric and intestinal pH and properly adhere to IEC-6 epithelial cells (which is an in vitro indicator of proper colonization in the gut) [51–53]. A major limitation to the present study was that the recovery of the probiotic bacteria was not assessed in feces, because most patients were unwilling to provide fecal samples. However, since the confounding factors were controlled (through keeping physical activity, anxiety, and nutritional intake constant
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throughout the study) and the ANCOVA analyses were used to compare the groups at the end of the study, it is most probable that the within-group changes and between-group differences are attributable to the treatment. On the other hand, the primary analyses for the present clinical trial [26] had shown positive effects of the treatment in patients; therefore, it is less likely that insignificant results in the present work are due to inefficient colonization of the administered probiotic bacteria. Along with the limitations of our study mentioned above, our work had a number of strength points; we considered the intakes of antioxidant nutrients to make sure that changes in dietary intake did not confound our results. Furthermore, evaluation of changes in physical activity and state-trait anxiety levels throughout the study course allowed for elimination of the possibility that alterations in these parameters had affected the oxidative status of the participants.
CONCLUSION We observed no significant effects of an 8week course of L. casei 01 supplementation on oxidative stress indices in women with RA. It is suggested that longer treatment durations be considered in future studies. Investigating the effects of other probiotic strains with confirmed antioxidant effects and at various dosages is encouraged.
ACKNOWLEDGMENTS We would like to thank the Management of Nutrition Faculty and the Drug Applied Research Center of Tabriz University of Medical Sciences, Tabriz, Iran, for supporting the laboratory tests. Special gratitude should go to the subjects who participated in the study, to Dr. Mohammad AsghariJafarabadi, who gave valuable advice on statistical analyses, and to Dr. Davoud Hassanzadeh, for helping us with preparation of the study supplements. The authors declare herein that they have no conflicts of interest.
FUNDING The present study was funded by the Research Vice Chancellor of Tabriz University of Medical Sciences, Tabriz, Iran.
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Received May 23, 2014; accepted August 25, 2014.
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