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the PJ group. Primary outcome data were assessed to identify any confounders and carry-over effects from period 1 to period 2 of the trial. If there were no confounders, repeated measures ANOVA was used to detect any differ- ence in UA glucuronide plasma levels between active (Mitopure) and comparator (PJ) over time. Absolute change was measured by comparing UA glucuronide plasma levels at the two time points. For the area under the curve (AUC) analysis, unpaired t -test was used to determine if there was a difference in AUC between Mitopure and PJ supple- mentation for UA glucuronide, UA sulfate, and parent UA. For microbiome sequencing results, statistical comparisons between groups were performed using the two-sided Mann – Whitney U test. To assess differences in the overall species community composition between the study groups (beta diversity), PERMANOVA was used. When perform- ing statistical testing on multiple hypotheses, the study used the BH method to control the FDR at a level of 10%.
and juice-based products (Supplementary Fig. 1). There was considerable variation, with some products having trace levels and others enriched in either EA or punicalagins. In all, 100% PJ was selected as the comparator as it showed a batch-to-batch consistency of the two-precursor compounds (71 mg punicalagins and 36 mg EA/8 fl . oz). Neither the supplements nor the PJ had detectable levels of UA in their matrix. The Mitopure powder reported a dose of 500 mg of UA within its matrix, with trace levels of precursor com- pounds from berry fruit powder added as fl avoring.
Compliance to study products and safety
Compliance was high with all participants consuming >95% of the study products. All participants completed the study and there were no drop-outs. No serious AEs were reported during the conduct of the study. There were 26 AE events reported following PJ intake and 15 AEs reported during the period of Mitopure intake (Supplementary Table 3). No particular differences were observed in the prevalence of any AE, most of which were mild and resolved during the study.
Results
Determination of dietary precursor levels in pomegranate-based supplements and juice
The majority of the study population failed to produce UA following dietary exposure to PJ
To identify the most suitable interventional product that contained dietary precursors and would lead to the genera- tion of UA, the study measured the levels of UA precursors (EA and punicalagins A and B) in the matrix of several commercial grade pomegranate extract dietary supplements
Only 12% of adults in the studied population had detectable levels of UA glucuronide at baseline, i.e., real-life free-living state (Fig. 2A). Subjects were categorized into three producer groups based on circulating UA glucuronide levels: non- producers (no detectable levels), low producers (<100 ng/mL),
A
B
C
T24h post intake
T6h post intake
T0 Baseline
32.70% 26.50% 40.80%
72.20% 23.70% 4.10%
87.80% 10.20% 2.00%
Total=100
Total=100
Total=100
No-Producer (no detectable UA-Glucuronide) Low-Producer (5-100ng/mL UA-Glucuronide) High-Producer (>100ng/mL UA-Glucuronide)
Fig. 2 Prevalence of UA producer status in the studied American population. Subjects were categorized into three producer groups based on circulating UA glucuronide levels: non-producers (no detectable levels), low producers (<100 ng/mL UA glucuronide), or high producers ( ≥ 100 ng/mL UA glucuronide). A At baseline only 12% subjects had detectable levels of UA glucuronide in circulation
with only 2% subjects classi fi ed as high producers. B Six hours fol- lowing dietary challenge with PJ approx. 28% subjects had detectable levels of UA glucuronide, with only 4% subjects high producers. C One day (T24 hours) after the PJ intake, approx. 40% of subjects had become high converters, whereas 60% still converted poorly or failed to convert the dietary precursors to UA.
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