A. Singh et al.
metabolites, UA glucuronide and UA sulfate, are the pre- dominant urolithin forms in circulation [5, 6]. UA has previously been shown to activate mitophagy, the recycling of defective cellular mitochondria, and to improve mitochondrial health in pre-clinical models of aging  and in a fi rst-in-human randomized, double-blind, placebo-controlled trial in elderly, sedentary subjects . The bene fi ts of UA have also been observed preclinically in several health indications, including neurodegenerative disorders, IBD, and metabolic dysfunction [8 – 10]. A few interventional studies have investigated the pre- sence of urolithin metabolites after intake of dietary fruits (strawberry and raspberry) and nuts (walnuts) in different study populations [11, 12]. Investigators have also exam- ined the association between different urolithin plasma pro fi les and health bene fi ts, such as lowering cardiovascular risk in overweight populations [2, 13, 14]. However, there are limited data in large and diverse age groups in healthy adult populations describing the frequency and extent of people ’ s natural ability to convert ET and EA into UA such as after drinking a glass of pomegranate juice (PJ) or eating the recommended quantities of nuts and berries. This report details the results from a clinical study in American adults (aged 20 – 80 years) that estimates the relative prevalence of UA producers and non-producers following a standardized dietary challenge with PJ. Meta- genomic pro fi les of the fecal samples of volunteers distin- guish the gut microbiome of UA producers from non- producers. The primary objective of the study was to com- pare the levels of UA and its conjugates (UA glucuronide being the main metabolite) in the circulation over time during a randomized, crossover study in which participants were orally administered either 100% PJ containing a natural pro fi le of UA precursor compounds to a nutritional product containing a calibrated dose of 500 mg of UA. The rationale of doing such a comparison was to document the variability and low levels of UA observed via natural exposure to diet rich in precursors vs. the uniform and consistent exposure obtained via direct supplementation with UA.
advertisements in local newspapers. From 136 screened participants, 100 successfully screened subjects were ran- domized on site to either the PJ or Mitopure intervention group (Fig. 1A). All subjects completed the study (last subject completed in February 2020) and there were no drop-outs. Among the randomized study participants, 68 were females and 32 were males (Supplementary Table 1.1). All were healthy and aged between 18 and 80 years, with a majority of either Caucasian, African-Amer- ican, or Hispanic ethnicity (Supplementary Table 1.2). Subjects were recruited to allow for a minimum of 20% representation in the following age groups: 18 – 40, 41 – 60, and 61 – 80 years. The remaining 40% were distributed among the different age groups. The average age of the population was 48.65 ±14.67 years (mean ± SD; Supple- mentary Table 1.3) with an average BMI of 29.76 ± 7.72 (mean ± SD; Supplementary Table 1.4). The study involved fi ve visits to the site over a 5-week period with a run-in phase of 3 weeks followed by the fi rst period of interven- tion. Following this fi rst period, a washout period of 8 – 14 days was implemented to ensure complete elimination of UA from circulation, as observed previously , after which subjects were administered the crossover dietary intervention (Fig. 1B).
Inclusion and exclusion criteria
To be eligible for inclusion, subjects needed to be in general good health de fi ned as the absence of any chronic ailments and as determined by the study medical doctor, and to have given voluntary, written, informed consent to participate. Subjects also needed to be willing to consume the investi- gational products, complete screening questionnaires, and complete all clinic visits during the study. Subjects were excluded if they had presence of any chronic medical con- ditions and clinically signi fi cant abnormal laboratory results at screening. Individuals deemed to be cognitively impaired and unable to give informed consent were excluded as well. Women who were pregnant, breastfeeding, or planning to become pregnant during the course of the trial, and subjects who had taken antibiotics within the previous 30 days of the study screening, were also excluded. Subjects were instructed to follow their usual diet and exercise routine and to not consume any other dietary supplements that could interfere with the assessment of the study products for the duration of the study. Subjects completed a food-frequency questionnaire (FFQ) during the screening visit to assess their dietary intake. The FFQ used for this purpose was the DHQ-III .
Study design and participant demographics
The trial was a single-center, two-period, crossover, ran- domized, open label study. It was approved by an inde- pendent institutional review board (Advarra IRB, Columbia, MD, USA) to be conducted at a clinical research site (Atlantia Food Trials, Chicago, IL, USA) and is registered as NCT04160312. Volunteers were recruited (from October 2019 to January 2020) through the clinical site database, social media platforms, general practitioners ’ of fi ces, and
Investigational products and randomization
Subjects who successfully passed the screening procedures were randomized on a 1:1 basis to receive either Mitopure
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