Nutrients 2023 , 15 , 2688
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Lifenol ® containing 100 µ g of 8-PN, 110 µ g of 6-PN, 1.25 mg of X, and 2.94 mg of IX (measured by LC-UV) mixed with maltodextrin (Roquette Fr è res, Lestrem, France), and filled in red opaque gelatin capsules size 0. Placebo capsules consisted of only maltodextrin within a similar type of capsules. 2.3. Bone Measurements by Dual-Energy X ray Absorptiometry Body composition was assessed with DXA. DXA examination, performed by the same health care professionals each visit, was conducted using the Lunar iDXA ME +210575 (GE Healthcare, Chicago, IL, USA). DXA was used to determine bone mineral density (BMD) and T score at each body site; DXA was also used to determine body composition (lean mass, fat mass, visceral fat, and fat percentage). Fracture risk assessment was determined using the FRAX tool (www.shef.ac.uk/FRAX, accessed on 7 December 2020) [26]. 2.4. Biomarkers of Bone Turnover and Biochemical Analysis Fasting blood samples collected in EDTA/heparin tubes were centrifuged at 3000 rpm at 4 ◦ C for 10 min within 40 min after collection. Samples were stored at − 80 ◦ Cuntil analysis. Plasma concentrations of osteocalcin and sclerostin were measured using an automated analyzer according to the manufacturer’s instructions (Multiplex Luminex ® As- says, Merck-Milipore, Burlington, MA, USA). Plasma concentrations of undercarboxylated osteocalcin (uOC) (Abbexa, Cambridge, UK), collagen type 1 cross-linked C -telopeptide (CTx) (Abbkine, Wuhan, China), procollagen type I N terminal propeptide (PINP) (Abbexa), human bone alkaline phosphatase (BALP) (MyBiosource, San Diego, CA, USA), tartrate- resistant acid phosphatase isoform 5b (TRAP5b) (MyBiosource), and BALP/TRAP5b ratio were measured using ELISA according to the kit manufacturer’s instructions. The manu- facturer supplied analytic variation coefficients were as follows: PINP and uOC (Abbexa): intra-assay CV < 10% and inter-assay CV < 12%; CTx, (Abbkine): intra-assay CV < 9% and inter-assay CV < 11%; BALP and TRAP5b (MyBiosource): intra-assay CV < 8% and inter-assay CV < 12%. Serum 25- hydroxyvitamin D (25-OH D3), plasma 17- β oestradiol, blood lipids (total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides), glucose homeostasis parameters (blood glucose, insulinaemia, HbA1c, and HOMA IR), and safety parameters were also measured. 2.5. Plasma and Urine Prenylflavonoids and Their Metabolites All prenylflavonoids (X, IX, 6-PN, and 8-PN) were measured both in plasma and urine in their unconjugated, glucuronide, and sulfated forms; each were expressed as a sum of the 3 different forms (i.e., total). Standard of X (purity: 99.6%), IX (purity: 99.6%), 8-PN (purity: 100%), and 6-PN (purity: 97%) were purchased from Phytolab (Vestenbergsgreuth, Germany). β -Glucuronidase enzyme from Escherichia coli , sulfatase enzyme from Helix pomatia , sodium phosphate, acetic acid, and sodium azide used for en- zymatic hydrolysis were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Since the concentrations measured in plasma and urine samples were very low, a calibration curve was prepared at very low concentrations ranging from 0.1 ng/mL to 5 ng/mL in methanol. Urine and plasma samples were stored at − 80 ◦ C until analysis; they were then prepared with an automated workstation (Beckman Coulter, Biomek NX, Brea, CA, USA) in random order. For the plasma samples, 100 µ L of plasma or enzymatic hydrolyzed plasma were loaded on Captiva EMR-lipid plate (Agilent Technologies, Santa Clara, CA, USA). An amount of 300 µ L of MeOH/ACN (50:50) were added and mixed at 700 rpm for 3 min. Samples were then eluted by positive pressure. An amount of 100 µ L of MeOH/ACN (50:50) were loaded on the cartridge once again and eluted by positive pressure. For the urine samples, 300 µ L of water were loaded on Captiva ND plate, and 100 µ L of urine or enzymatic hydrolyzed urine were added and mixed at 700 rpm for 1 min. Samples were then eluted by positive pressure and analyzed by LC-HRMS. Enzymatic hydrolyzed urine and plasma samples were prepared with 10 mg of enzyme diluted in 10 mL sodium
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