Direct supplementation with Urolithin A overcomes limitations of dietary exposure and gut microbiome variability in healthy adults to achieve consistent levels across the population

Abstract

Background: Urolithin A (UA) is produced by gut microflora from foods rich in ellagitannins. UA has been shown to improve mitochondrial health preclinically and in humans. Not everyone has a microbiome capable of producing UA, making supplementation with UA an appealing strategy.

Objective: This is the first detailed investigation of the prevalence of UA producers in a healthy population and the ability of direct UA supplementation to overcome both microbiome and dietary variability. Dietary intake of a glass of pomegranate juice (PJ) was used to assess UA producer status (n = 100 participants) and to characterize differences in gut microbiome between UA producers from non-producers.

Methods: Subjects were randomized (1:1) to either PJ or a food product containing UA (500 mg). Prevalence of UA producers and non-producers were determined in the PJ group. Diet questionnaires and fecal samples were collected to compare differences between UA producers and non-producers along with plasma samples at different time points to assess levels of UA and its conjugates between the interventions.

Results: Only 12% of subjects had detectable levels of UA at baseline. Following PJ intake ~40% of the subjects converted significantly the precursor compounds into UA. UA producers were distinguished by a significantly higher gut microbiome diversity and ratio of Firmicutes to Bacteroides. Direct supplementation with UA significantly increased plasma levels and provided a >6-fold exposure to UA vs. PJ (p < 0.0001).

Conclusions: Differences in gut microbiome and diet that dictate natural exposure to UA can be overcome via direct dietary UA supplementation.

Conflict of interest statement

The authors declare the following competing interests: AS, DD, PAA, WB-B, and CR are employees; PA and CR are board members; and JA and PA are members of the Scientific Advisory Board of Amazentis SA, who is the sponsor of this clinical study.

© 2021. The Author(s).

Figures

Fig. 1. A two-period, crossover, randomized trial study design in healthy adults comparing PJ dietary challenge to Mitopure supplementation.

A The corresponding CONSORT diagram is represented. A total of 136 subjects were screened in the study, following which randomization occurred (n = 100) to one of the two study interventions in sequential manner. Following a washout period of 8–14 days, the crossover period occurred in which subjects took the second intervention for comparison of bioavailability. All subjects completed the study and there were no drop-outs or major protocol violations resulting in all data being analyzed. B Simplified schema of the clinical study design. In total there were five study visits: Visit 1 (screening), Visit 2 (randomization, blood collection at baseline (T0) and 6 h following intake (T6)), Visit 3 (blood collection 24 h after intake of 1st intervention (T24)). The three first visits were followed by a washout period of 8–14 days after which the crossover intervention occurred with the corresponding blood draws (Visits 4 and 5).

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 (A At baseline only 12% subjects had detectable levels of UA glucuronide in circulation with only 2% subjects classified as high producers. B Six hours following 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.

Fig. 3. Gut microbiome differences between UA producers and non-producers.

A Boxplots showing differences in metagenomics species (MGS) for richness (left panel) and Shannon diversity (right panel) between groups with no, low, and high UA producer status. All groups were compared pairwise by Mann–Whitney U test (N = 99). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; *****p ≤ 0.00001. B Principal coordinate analysis (PCoA) based on Bray–Curtis dissimilarities among samples, calculated based on the MGS abundances. Samples are color coded by the UA producer status. Each sample is connected to its group centroid by a thin segment line. The ellipses cover two standard errors of the mean of the group centroids, i.e., they illustrate the certainty of the group centroid positions. The x- and y-axis labels indicate the microbial variance explained by the first two principal coordinates. C Relative abundance (in %) of phyla Firmicutes and Bacteroidetes that significantly differed in abundance in producer of UA compared with no producer. Boxes represent interquartile range (IQR), with the inside horizontal line representing the median. Whiskers represent values within 1.5× IQR of the first and third quartiles. D Firmicutes/Bacteroidetes ratio (F/B ratio) for each group shown as median (IQR). **p ≤ 0.01 (Mann–Whitney U test).

Fig. 4. Mitopure supplementation delivers significantly higher plasma UA levels compared to PJ.

Pharmacokinetic profiles at T0, T6 (6 h), and T24 (24 h) and mean absolute change in levels from T0 to T24 (primary outcome of study) between the two interventions of PJ and Mitopure supplementation for UA glucuronide (A, B), UA sulfate (C, D), and parent UA (E, F) showing significantly higher plasma levels of UA and its metabolites with Mitopure supplementation compared to PJ dietary challenge (N = 100). All data are analyzed using a repeated measure ANOVA (A, C, E) and an unpaired t-test (B, D, F).

Fig. 5. Mitopure supplementation delivers >6-fold higher exposure to UA compared to PJ and achieves consistent levels across the adult population.

Mean incremental area under the curve (iAUC) within a day following intake of Mitopure compared to the consumption of a glass equivalent of 100% PJ, for UA glucuronide (A), UA sulfate (B), and the parent UA (C) showing higher exposure to UA and its metabolites with Mitopure compared to PJ (N = 100). All data are expressed as mean ± SEM and analyzed using an unpaired t-test. Correlation of UA glucuronide levels across different age groups at 6 h following either PJ intake or direct UA supplementation (D). Frequency distribution of UA glucuronide levels observed across the population following both PJ and direct UA supplementation at 6 h (E) and 24 h post intake (F).