A. Rehman et al.
The Journal of Nutrition xxx (xxxx) xxx
resulting bins were analyzed using MetaboAnalyst v4.0 [36] after being divided into 2 groups, i.e., subjects who had received Fruit fl ow (group 1, visit 5; group 2, visit 3) and subjects who had not (group 1, visit 2, 3, and 4; group 2 visit 2, 4, and 5). All data were normalized (quantile normalization) and auto-scaled (Pareto scaling) before univariate (t-test) and multivariate analysis (PLS-DA). The Q2 and R2 values for Partial Least Squares Discriminant Analysis (PLS-DA), as well as the Variable Importance in Projection (VIP) plots, were determined by MetaboAnalyst following an established work fl ow[37]. A paired Wilcoxon test was used for microbial alpha diversity to determine the statistical signi fi cance between and within groups. For beta diversity, Permutational Multivariate Analysis of Variance (PERMANOVA) was used to quantify differences within and between groups (Fruit fl ow compared with placebo) using 9999 permutations. This test evaluates whether groups or time points signi fi cantly affect the overall gut microbiota composition and structure. Within and between-group changes in the relative abundance of microbial taxa (i.e., the difference in outcome between the Fruit fl ow and placebo group) was analyzed using paired Wilcoxon test followed by multiple testing correction with Benjamini-Hochberg. In addition, for between- group changes, raw abundance counts were Centroid Log Ratio normalized to ensure that the data is scale-invariant and sub- compositionally coherent. Finally, the effect size differences for each taxon were calculated using the cohens_d function (https://easystats.github.io/effectsize/), an R-based package.
but lumpy), Type 3 (like a sausage but with cracks on its surface), Type 4 (like a sausage or snake, smooth and soft), Type 5 (soft blobs with clear-cut edges), Type 6 ( fl uffy pieces with ragged edges, a mushy stool), Type 7 (watery, no solid pieces, entirely liquid) [25]. Gastrointestinal symptoms were assessed by a 6-point scale using the GSRS utilizing a 7-point rating scale, depending on the intensity and frequency of gastrointestinal symptoms experienced during the previous wk. A high score indicated more inconvenient symptoms. Investigational product Fruit fl ow was commercially produced by DSM Nutritional Products, Basel, Switzerland, in powder format. The composition of Fruit fl ow has been described previously [32] with a standard 150 mg dose delivering up to 9 mg nucleoside derivatives, up to 10 mg simple phenolic conjugates (e.g., chlorogenic acid, other caffeic or phenolic acid derivatives), and up to 7 mg fl avonoid derivatives, of which at least 2.4 mg are quercetin derivatives. The ef fi cacious range for Fruit fl ow to affect platelet aggregation and thrombin generation capacity lies between 75 and 300 mg [33]. Maltodextrin was used as a placebo control (Essential Nutrition Ltd., Brough, UK). All supplements were encapsulated using size 00 Vegecaps (LGA, La Seyne-sur-Mer, France), and the fi nal weight of each capsule was 500 mg (weight of Fruit fl ow plus weight of tapioca starch fi ller). Participants were instructed to consume 2 capsules orally (either placebo or Fruit fl ow) in the morning at breakfast along with a glass of water resulting in a total of 300 mg Fruit fl ow per d for a 28-d intervention period. Sample size calculation and statistical analysis The sample size was determined based on the fi ndings from previous studies with nutritional interventions to reduce plasma TMAO [9, 34, 35]. For a power of 80%, the signi fi cance level of 5%, and an expected effect size [mean (SD)] of 0.5 for Fruit- fl ow compared with placebo, it was calculated that 34 partici- pants were required. To account for potential losses to follow-up, 40 participants were enrolled. The effectiveness of the wash-out was assessed using paired samples t-tests comparing the baseline of phase 1 (visit 2) with the baseline of phase 2 (visit 4). There were no statistically sig- ni fi cant differences between any parameters at phase 1 baseline and phase 2 baseline for the Fruit fl ow or placebo group. As a result, the Fruit fl ow group is a combination of group 2 – phase1 data (visit 2-visit 3) and group 1 – phase 2 data (visit 4-visit 5), whereas the placebo group is a combination of group 1 – phase1 data (visit 2-visit 3) and group 2 – phase 2 data (visit 4-visit 5). SPSS IBM V26.0 and R software were used to analyze all data. For the ef fi cacy analysis, paired t-tests (or Wilcoxon signed rank tests) were used to determine 1) whether there was a statistically signi fi cant within-group change from baseline to end of the intervention in either the Fruit fl ow group or the placebo group or 2) whether there was a statistically signi fi cant between-group difference (i.e., differences in changes between groups). All data are reported as means SE. All tests were 2-tailed, and differ- ences were considered statistically signi fi cant at P 0.05. For untargeted metabolomics, all spectra were collected and processed using ACD Labs 2012. All spectral regions containing NMR features were group processed by Intelligent Bucketing (Bucket width ¼ 0.02 ppm, width looseness ¼ 50%). The
Results
Study population The study population consisted of 40 overweight and obese adults. Three participants self-withdrew after randomization whereas the remaining 37 completed the study. An independent committee subsequently evaluated all participants case by case before unblinding the data to determine inclusion in the per- protocol analysis. Fifteen subjects were excluded from the analysis becauseofusingconcomitantmedicationssuchasantibiotics( n ¼ 9) and investigational product (IP) compliance below 80% and/or missing critical variables (because of missing blood and stool sam- ples, n ¼ 6);therefore,theper-protocoldataanalysiswasbasedon22 subjects ( see Supplemental Figure 2 for the CONSORT (Consoli- dated Standards of Reporting Trials) fl ow diagram and Supple- mentalTable1forsubjectcharacteristicsofthe fi nal22participants)
TMAO
Fasting plasma and urine TMAO. Fruit fl ow reduced fasting urine, but not fasting plasma, TMAO concentrations when compared with placebo ( P 0.05, Figure 1A, B). When comparing baseline to end of the intervention time points within each group, Fruit- fl ow, but not placebo, signi fi cantly reduced both fasting plasma ( 1.51 μ M, P 0.05) and fasting urine TMAO concentrations ( 19.09 μ M, P 0.01, Figure 1A and B). Postprandial plasma and urine TMAO during the egg challenge. The egg challenge was performed only in a subgroup of participants either at visit 3 or 5 to compare postprandial changes in plasma TMAO between groups. Overall, there was a large variation
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