Direct supplementation with Urolithin A overcomes limitatio…

Direct supplementation with Urolithin A overcomes limitations of dietary exposure and gut microbiome. . .



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 Simpli fi ed schema of the clinical study design. In total therewere fi ve 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 fi rst 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).

Assessed for eligibility (n=136)


Excluded (n=36) Not meeting inclusion criteria (n=26) Declined to participate (n=6) Lost to follow-up (n=4)

Randomized (n=100)


Allocated to 100% Pomegranate Juice (n=50)

Allocated to Mitopure (Urolithin A) intervention (n=50)



Allocated to 100% Pomegranate Juice (n=50)

Allocated to Mitopure (Urolithin A) intervention (n=50)


Analysed (n=100)

Analysed (n=100)


Day 1

Day 0

Day (10 to 16)

Day (-21 to -2) -

Day (9 to 15)

Group 1 (n=50)

Run-in phase


Group 2 (n=50)

Visit 5:

Visit 3: Blood (AM)

Visit 2: Randomisation

Visit 4:

Visit 1: Screening

Blood (AM) Blood (PM)

Blood (PM) Blood (AM)

Blood (AM)

Intervention period 1

Intervention period 2

100% compliance. Mitopure powder was admixed into a vanilla- fl avored commercial yogurt (4 fl . oz) and the PJ was served chilled. Participants were asked to consume the investigational product within 10 min. The time of the fi rst sip of PJ and spoonful of yogurt intake containing admixed Mitopure was recorded as the time of product ingestion in the study database.

(500 mg UA) oral supplementation or 8 fl . oz (~240 mL) of PJ. Natural precursors to UA (EA and punicalagins) and UA levels were assessed in each batch of the PJ and Mitopure administered using calibrated and validated HPLC methods. Randomization was carried out by a study- independent statistician using a computer-generated pro- gram to create a block randomization list. Each subject was assigned a unique randomization number. Participants were randomized to one of the following sequence groups as follows:

Blood and fecal samples collection

Plasma and dried blood spot (DBS) samples were collected from all subjects at baseline (T0), at 6 h (T6), and 24 h (T24) after the intake. During the screening visit, approxi- mately 16 mL of blood was collected to assess clinical, laboratory, and hematology parameters. Following that, at each visit and time point, an 8 mL blood sample was drawn into K2-EDTA coated tubes (Supplementary Table 2). The blood samples were gently inverted several times to ensure complete mixing with the anticoagulant. The exact time of sample collection was recorded in the study database. Within 30 min of collection each blood sample was

Group 1: Mitopure – Pomegranate Juice Group 2: Pomegranate Juice – Mitopure

The investigation products were the Mitopure powder, a fruit- fl avored powder containing 500 mg of pure UA and ingredients that include blueberry, raspberry fruit powder, pomegranate natural fl avoring, and fi ber and 8 fl . oz (~240 ml) of a commercially available 100% PJ (POM Wonderful, Los Angeles, CA, USA). Intake happened onsite and was supervised by the research staff to ensure

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