The Effects of Synbiotic Supplementation on Carotid Intima-Media Thickness, Biomarkers of Inflammation, and Oxidative Stress in People with Overweight, Diabetes, and Coronary Heart Disease: a Randomized, Double-Blind, Placebo-Controlled Trial
Authors: Fariba Raygan
Alireza Farrokhian
Maryam Tajabadi-Ebrahimi
Ali Akbar Karami
Zatollah Asemi
Mehran Sharifi Esfahani
Ali Soltani
Journal: Probiotics and Antimicrobial Proteins (2019)
Discipline: MICROBIOLOGY
Keywords: Carotid intima-media thickness; Coronary heart disease; Inflammation; Oxidative stress; Synbiotic supplementation
Affiliations: Department of Cardiology, School of Medicine, Kashan University of Medical Sciences, Kashan, I.R., Iran., Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan, I.R., Iran., Science Department, Science Faculty, Islamic Azad University, Tehran Central Branch, Tehran, Iran., Department of Oncology, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran., Department of Urology, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran., Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan, I.R., Iran. .

Abstract:  Synbiotics are known to exert multiple beneficial effects, including anti-inflammatory and antioxidant actions. The aim of this study was to evaluate the effects of synbiotic supplementation on carotid intima-media thickness (CIMT), biomarkers of inflammation, and oxidative stress in people with overweight, diabetes, and coronary heart disease (CHD). This randomized, double-blind, placebo-controlled trial was conducted and involved 60 people with overweight, diabetes, and CHD, aged 50-85 years old. Participants were randomly allocated into two groups to take either synbiotic supplements containing three probiotic bacteria spices Lactobacillus acidophilus strain T16 (IBRC-M10785), Lactobacillus casei strain T2 (IBRC-M10783), and Bifidobacterium bifidum strain T1 (IBRC-M10771) (2 × 109 CFU/g each) plus 800 mg inulin or placebo (n = 30 each group) for 12 weeks. Fasting blood samples were taken at baseline and after the 12-week intervention period to determine metabolic variables. After the 12-week intervention, compared with the placebo, synbiotic supplementation significantly reduced serum high-sensitivity C-reactive protein (hs-CRP) (- 3101.7 ± 5109.1 vs. - 6.2 ± 3163.6 ng/mL, P = 0.02), plasma malondialdehyde (MDA) (- 0.6 ± 1.0 vs. - 0.1 ± 0.3 μmol/L, P = 0.01), and significantly increased nitric oxide (NO) levels (+ 7.8 ± 10.3 vs. - 3.6 ± 6.9 μmol/L, P &lt; 0.001). We did not observe any significant changes of synbiotic supplementation on other biomarkers of oxidative stress and CIMT levels. Overall, synbiotic supplementation for 12 weeks among people with overweight, diabetes, and CHD had beneficial effects on serum hs-CRP, plasma NO, and MDA levels; however, it did not have any effect on other biomarkers of oxidative stress and CIMT levels. 

Retracted: No

PubPeer comments:
 The authors state in this article that  Based on the study provided by Van De Weert and Stella [29], the Sternu2013Volmer quenching rate constant (Kq) and apparent binding constant (Ka) were consequently obtained by linear regressions of the Stemu2013Volmer equation and double log Stemu2013Volmer equation (Figure S3) and shown in Table 4. Reference 29 is a paper of mine, and sadly it appears the authors did not fully understand the paper. In Van de Weert Stella we point out numerous pitfalls with the double log Stern-Volmer equation, and it appears the authors have just simply ignored all of those. The first pitfall is the impact of the inner-filter effect: changes in absorbance upon addition of the ligand. This can cause quenching without an actual interaction. Note that this issue also applies to the ordinary Stern-Volmer equation. It is not made clear in the article that the authors corrected for this, or even looked whether it may have played a role. A second pitfall is that the equation assumes the complex is non-fluorescent. This assumption needs to be shown to be true, as it is unlikely that binding of a ligand completely quenches the fluorescence of all Trp residues in a protein. Finally, we point to the issue of added vs free ligand concentration, but this may not be an issue here. Indeed, we also point to the problematic interpretation of  n in this equation, and the authors at least mention this aspect, but this does not mean the other issues can just be overlooked. A final issue is that the authors use the value of k(q) (and note this should be a small k, as it is a rate constant) to argue the quenching is  static . This once again does not follow from our paper, as we explain that this dichotomy only works for two small molecules interacting through either collisions or through formation of a ground-state complex. In proteins quenching mechanisms can include various other factors, such as energy-transfer. The latter is, by IUPAC definition, a dynamic quenching process, but involves direct binding, rather than collisions. , 
 #1 First of all, I feel exciting to communicate with the author of our reference. In deed, we are not expert in fluorescence, therefore may misunderstanded the meaning expressed by your published work. I just tracked back to your paper and also find out the conclusion is mainly about the prerequisite of fluorescence analysis. Thanks for your comments. As you mentioned above:  In proteins quenching mechanisms can include various other factors, such as energy-transfer . It will be very pleased if you could provide some suggestions about using fluorescence to characterize the interaction between proteins and lingards. For example, during fluorescence analysis, how to eliminate other impacts (such as energy-transfer) from protein-lingard interaction? Is the fluorescence analysis approperate for studying protein-lingard interaction? Or, for the protein-lingard interaction study, other methods (such as NMR ITC XRD other than fluorescence) could be a better chioce? Thanks in advance because this is very important for our future study. :) , 
 #2 Thank you for your reply on this. In principle fluorescence is a powerful method to look at ligand binding, it is mostly a matter of taking care of the pitfalls (such as those I mentioned) and use equations that correct for these. For example, it does not matter what the mechanism of quenching is upon binding - except of course the inner filter effect - if one wants to determine the binding constant. Just make sure to use the right equations, even if one sometimes has to make some assumptions (like 1-to-1 binding). If fluorescence lifetime measurements are possible in your lab, you can also use these to get some idea on the quenching mechanism. Finally, in some cases the ligand has a fluorescent signal that can be used to characterize binding. This is much better than using the intrinsic fluorescence of the protein, as the latter will only change if the ligand binds closely to the fluorophore(s). Notably, it is not perfect. ITC is much more powerful for a proper binding analysis, but also has some pitfalls that are not always clear. NMR may be useful as well, but is often a bit slower. I recommend you read this book: https: www.springer.com gp book 9781071611968 or earlier versions of this book. , 
 Thanks a lot, we really appreciate for your help and suggestions on fluorescence and protein ligand binding. ,