Nutrients 2023 , 15 , 3466
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ELLA Protein Simple plex (BioTechne, Dublin, Ireland) was used to analyze BDNF (#898072, detection limit 9.4–36,000 pg/mL), fractalkine (#898143, detection limit 56.8–86,710 pg/mL), TGF- β 1 (#898477, detection limit 20.8–12,684 pg/mL), and galectin-3 (#898088, detection limit 3.11–4741 pg/mL) in serum. Plasma levels of tryptophan and L-kynurenine were measured with commercially available ELISA kits (ImmuSmol, Bordeaux, France). The L-kynurenine kit (BA E-2200) had a detection limit of 45.7 ng/mL, and the tryptophan kit (BA E-2700) had a measuring range of 0.73–250 µ g/mL. Hs-CRP was measured by the medical service at Skåne University Hospital (University and regional laboratories in Region Skåne, Sweden) using an immune turbidimetric analysis with a detection range between 0.16 and 10 mg/L. 2.6. Safety Blood samples for safety parameters (clinical biochemistry and hematology) were collected at visit 1 (screening), visit 2 (baseline), and visit 5 (12 weeks). The samples were analyzed by Eurofins Biomnis, Dublin, Ireland. Safety was also assessed via monitoring of adverse events (Aes) and physical examinations. The subjects were also questioned about their medication history during the screening visit and thereafter queried at each visit about the use of any concomitant medications. 2.7. Statistical Methods As this was a novel exploratory study, there was no pre-existing data to complete an a priori sample size calculation. The results presented in this paper are for the intention to treat (ITT) population if not otherwise indicated. ITT includes all randomized subjects with at least one reported consumption of any of the products. The per protocol population (PP) includes all ran- domized subjects with complete data for the primary outcomes, and a minimum of 80% reported compliance in terms of the amount of product consumed with no major protocol deviations. All subjects, who took at least one dose of the study product, were included in the safety population. All analyses were two-sided at a 5% significance level. However, since it is an ex- ploratory study, some p -values of >0.05 were also reported. For each endpoint, if there was no statistically significant difference(s) at baseline between the two groups, a mixed two-way between–within groups ANOVA was used to determine whether there was a statistically significant change over time in the endpoint between the two groups from baseline (visit 2) to the end of the intervention (visit 5). For some variables, outliers were identified (data that were greater than 1.5 box lengths from the edge of the boxplot) and re- moved from the analysis. Sensitivity analyses were furthermore conducted using two-way between–within groups ANOVA to determine whether there was a statistically significant change over time in the endpoint between the two groups from baseline (visit 2) to week 4 (visit 3) to week 8 (visit 4) until the end of the intervention (week 12; visit 5). The difference between the groups in terms of change from baseline to each timepoint (week 4, week 8, and week 12) separately was also evaluated using an unpaired t -test. The non-parametric Mann-Whitney U Test was implemented as an alternative to parametric estimates when the assumptions of those methods were in doubt, or when parametric inference was impossible. An analysis of the change from baseline within a group was conducted using paired t -test or the non-parametric test Wilcoxon Signed-Rank test or Friedman’s test. If the data were categorized, McNemar’s test was used to determine if there was a statistically significant within-group change in terms of the proportion of subjects in each category from baseline to the end of the intervention, and chi-square analysis was used to evaluate differences between the groups at each time point. All analyses were conducted using the SPSS IBM V 28.0.
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