The effects of probiotic supplementation on metabolic status in type 2 diabetic patients with coronary heart disease

This study was a randomized, double-blind, placebo-controlled trial registered in the Iranian registry of clinical trials (http://​www.​irct.​ir: IRCT2017082733941N5) which performed at a cardiology clinic affiliated to Kashan University of Medical Sciences (KAUMS), Kashan, Iran, from October 2017 through January 2018. Inclusion criteria were as follows: patients with T2DM, aged 40–85 years old with 2- and 3-vessel CHD. T2DM was diagnosed based on the criteria of the American Diabetes Association [13]. Furthermore, the diagnosis of CHD was conducted following the American Heart Association guideline [14]. Exclusion criteria included consuming probiotic and/or synbiotic within 3 months prior to the intervention, taking prebiotic, antioxidant and/or anti-inflammatory supplements such as vitamin E, vitamin C and omega-3 fatty acids, taking antibiotics, having an acute myocardial infarction, a cardiac surgery in the past 3 months, renal or hepatic failure.

The research was done following the Declaration of Helsinki principals. The protocol of this study was approved by Research Ethics Committee, KAUMS, and Iran. Written informed consent was taken from all participants.

Initially, we conducted a stratified randomization for all participants according to age, BMI, gender, the dosage and kind of medications, in order to decrease potential confounding effects. Then, participants in each stratum were randomly allocated into two treatment groups to take either probiotic supplements including Bifidobacterium bifidum 2 × 10⁹, Lactobacillus casei 2 × 10⁹, Lactobacillus acidophilus 2 × 10⁹ CFU/day (n = 30) or placebo (n = 30) for 12 weeks. Color, shape, size, and package of placebos and probiotics capsules were identical and made by Tak Gen Zist Pharmaceutical Company (Tehran, Iran). Randomization was conducted using computer-generated random numbers. Randomization and allocation were concealed from the investigators and participants until the final analyses were completed. The randomized allocation sequence, enrolling participants and allocating them into intervention groups were performed by a trained staff at cardiology clinic. At the beginning of the study, patients were requested to maintain their regular diet and levels of physical activity throughout the period of the trial. Compliance rate regarding the consumption of placebos and probiotic supplements was determined by examining the returned capsule containers. All patients completed 3-day dietary records at baseline, week 1, 4, 8 and 12 of treatment. To determine nutrient intakes of participants according to 3-day food records, we used Nutritionist IV software (First Databank, San Bruno, CA). Physical activity was quantified as metabolic equivalents (METs) in hours per day [15]. Anthropometric measures (Seca, Hamburg, Germany) were measured at baseline and end of the intervention at the cardiology clinic by a trained staff. Nutritionist also was blinded to the randomization assignments.

Insulin metabolism was considered as primary outcome, however lipid profiles, biomarkers of inflammation and oxidative stress were considered as secondary outcomes. Fasting blood (10 mL) was taken at baseline and after the 12-week intervention at Kashan reference laboratory, Kashan, Iran. Insulin levels were measured using an ELISA kit (DiaMetra, Milano, Italy) with inter-assay and intra-assay coefficient variances (CVs) of lower than 5%. The homeostasis model of assessment-insulin resistance (HOMA-IR) and the quantitative insulin sensitivity check index (QUICKI) were calculated according to the standard formulas [16]. Enzymatic kits (Pars Azmun, Tehran, Iran) were used to determine fasting plasma glucose (FPG) and lipid profiles with inter- and intra-assay CVs of lower than 5%. Hs-CRP levels were measured using an ELISA kit (LDN, Nordhorn, Germany) with inter- and intra-assay CVs of lower than 7%. Nitric oxide (NO) was measured by Griess assay [17], total antioxidant capacity (TAC) the method reported by Benzie and Strain [18], total glutathione (GSH) by Beutler method [19], and MDA concentrations by the spectrophotometric test with inter- and intra-assay CVs of lower than 5% [20]. Systolic (SBP) and diastolic blood pressure (DBP) was measured using a sphygmomanometer (ALPK2, Zhejiang, China). Blood pressure values were reported in millimeters of mercury (mmHg).

We calculated sample size using the formula suggested for randomized clinical. Type one (α) and type two errors (β) were defined as 0.05, and 0.20 (power = 80%), respectively. According to the previous trial [21], we used 1.61 as the SD and 2.30 as the mean change (d) of HOMA-IR. Based on the formula, 25 participants were required in each group; after allowing for 5 dropouts in each group, the final sample size was 30 participants per intervention group.

A few studies have evaluated the effects of probiotic supplementation on glycemic control and lipid profiles among diabetic patients; however this data is scarce among diabetic patients with CHD. In a meta-analysis conducted by Yao et al. [22], probiotic ingestion significantly reduced HbA1c and insulin levels in patients with T2DM, but did not affect their lipid profiles. In addition, Taylor et al. [7]. found that probiotic supplementation for 6–8 weeks resulted in a significant reduction in insulin resistance in pregnant women with GDM, but did not influence their lipid profiles and fasting glucose levels. Furthermore, it was reported that gut microbiota contributes to glucose homeostasis through different bacterial metabolites [23]. Previous meta-analyses revealed that probiotic supplementation decreased insulin resistance and glycated hemoglobin levels [24, 25]. In contrast, such beneficial effects were not reported by others [26‐29]. Insulin resistance and dyslipidemia in patients with T2DM increase the risk of microvascular complications and cardiovascular mortality [30]. Therefore, probiotic administration might be useful to decrease diabetic complications, due to its glucose-lowering effect. Improved glucose homeostasis parameters, HDL- and total-/HDL-cholesterol ratio by probiotics among diabetic patients with CHD may be related to their role in decreasing inflammatory cytokines and suppressing the nuclear factor-κB pathway [31], their impact on gene expression [12] and the activation of gut microbiota-short chain fatty acids (SCFA)-hormone axis [32].

We have previously shown that taking probiotics by patients with major depressive disorder for 8 weeks improved hs-CRP and GSH concentrations, but did not influence TAC levels [28]. Furthermore, a significant reduction in hs-CRP levels was seen after taking probiotic supplements for 48 weeks by HIV-infected individuals [33]. On the other hand, no significant changes in MDA and TAC levels were found following probiotic capsule supplementation containing Lactobacillus casei (10⁸ CFU/g) for 8 weeks in patients with rheumatoid arthritis [34]. Diabetes is correlated with enhanced concentrations of vascular inflammation and oxidative stress parameters [35]. Cell culture studies suggest that elevated pro-inflammatory markers may cause a reduction in GSH synthesis [36, 37]. Discrepancies in these findings may be explained through the differences in study design, characteristics of study populations, probiotic species, strains and formulations available, and duration of the intervention. Probiotics ingestion may decrease inflammation and oxidative stress through increased production of SCFA in the colon, improved activity of glutamate-cysteine-ligase activity (GCL) and increased gene expression of GCL subunits [38].

This study had few limitations. In the current study, fecal bacterial loads were not measured before and after probiotic consumption. Also, there was no chance to evaluate gene expression of lipid, insulin, inflammation and oxidative damage in diabetic patients with CHD.