Nutrients 2023 , 15 , 2688
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phosphate buffer (100 mM, pH = 6.8) for β -Glucuronidase and in 10 mL sodium acetate buffer (100 mM, pH = 5) for sulfatase enzyme. An amount of 60 µ L of this solution were mixed with 60 µ L of plasma or urine and incubated during 1 h at 37 ◦ C. Noting the difference between the concentrations measured before and after enzymatic hydrolysis provides the ability to calculate the concentrations of the glucuronide/sulfated forms. Liquid chromatography was performed on a UHPLC Thermo Vanquish (Thermo Scientific, Karlsruhe, Germany) in reverse phase mode with an Accucore RP-MS column (150 × 2.1mm, 2.6 µ m, Thermo Scientific) using solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in ACN). The elution gradient started at 5% B for 1 min, followed by a linear gradient rising to 90% B during 9 min. The mobile phase remained at 90% B for 5 min and then returned to initial condition after 1 min. The column was equilibrated for 4 min in initial conditions (5% B) prior to the next injection, for a total run time of 20 min. The flow rate was 0.5 mL/min, and the injection volume was 2 µ L. The column was heated at 30 ◦ C to ensure a stable column temperature and a better repeatability between runs, and the autosampler temperature was maintained at 6 ◦ C. The UHPLC system was coupled to an Orbitrap Q-Exactive Focus mass spectrometer (Thermo Scientific, Germany), and analyses were performed using an electrospray interface in negative mode, in full scan with a resolution of 35,000 FWHM in the scan range of m / z 80–1000. ESI parameters were as follows: heater temperature 300 ◦ C, capillary temperature 350 ◦ C, sheath gas 55 (arbitrary units), auxiliary gas 15 (arbitrary units), S-Lens 50 V, spray voltage: 3.5 kV in ESI-. Quan Browser software (Thermo Scientific) was used for quantification and the 4 targeted compounds were extracted with a mass window width of 5 ppm. This analytical method was validated according to internal guidelines and specificity, linearity, repeatability, and limit of quantification (LOQ) are listed in Table S1. Accurate mass used for the four compounds of interest is also listed and specificity was tested by checking that matrices and diluent did not interfere with the analyte masses and retention time. Repeatability was established by analyzing 6 samples of standards solution in diluent and 6 spiked plasmas at 20 ng/mL. The relative standard deviation (RSD) of this analytical method ranged from 3.2 to 4.7% for standards solution in diluent and from 2.5 to 3.8% in spiked plasma according to the compounds (Table S1). Calibration curves also displayed satisfactory linearity with R 2 greater than 0.99 for all compounds, and LOQ were established using a signal-to-noise ratio above 10. 2.6. Dietary Intake, and Physical Activity Level, and Quality of Life Dietary habits, using the EPIC-Norfolk Food Frequency Questionnaire (FFQ; https:// www.epic-norfolk.org.uk/, accessed on 7 December 2020), were evaluated at baseline and after 48 weeks. Data were entered into the Nutritics software version 5.61 (Nutritics, Dublin, Ireland), and average daily intake of total energy, fat, carbohydrate, protein, fiber, calcium, vitamin D, and isoflavones were derived. Physical activity level was registered using the self-reported level of Physical Activity Scale for the Elderly (PASE) [27]. The PASE total score range from 0–400, where higher scores reflect a higher activity level. Health-related quality of life was measured by measuring the means scores of the Short Form 36 (SF-36) [28]. The SF-36 is divided into eight sub-scales (physical function, role limitations-physical, bodily pain, general health, vitality, social function, role limitations- emotional, and mental health). The measurement is scored on a 0–100-point scale for each sub-scale; the higher the score, the more positive the health status.
2.7. Gut Microbiome Analysis 2.7.1. Fecal Samples Collection and DNA Extraction
Fecal samples were collected at baseline, at 24 weeks, and at 48 weeks. Participants were provided stool collection kits and instructed to collect an at-home sample within 48 h of their next research visit. The fecal sample was collected using a collection vial and then placed immediately in the home freezer ( − 20 ◦ C) before being brought to the clinic in a
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