Nutrients 2020 , 12 , 141
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T0 FMD. At 60 min (T1), 120 min (T2), and 180 min (T3) after the start of the citrus drink consumption, FMD scans and blood sample collections were performed. 2.3. Flow ‐ Mediated Dilation Ultrasound FMD scanning procedures were strictly standardized and quality ‐ controlled as described in extenso elsewhere [34,35]. In short, prior to the start of the study, two research technicians on site (Atlantia Food Clinical Trials CRO, Cork, Ireland) were trained and certified according to pre ‐ set criteria of the FMD core lab (Imagelabonline & Cardiovascular, Erichem, The Netherlands). All scans were performed with an Ultrasonix Sonix SP ultrasound instrument equipped with an L14 ‐ 5MHz vascular transducer and fixed presets throughout the study (Analogic Corporation, Peabody, MD, USA). The examinations took place in a quiet room at temperatures between 20 °C and 26 °C and dimmed lighting. For each subject on both test days, initial and follow ‐ up scans were performed by the same technician. Subjects comfortably reclined for 20 min prior to scans. During visits, the subjects’ right arm rested in the arm ‐ supporting cups of the probe ‐ holder arm rest in a fixed position during the 9 ′ scan procedures. The brachial artery was scanned longitudinally to obtain high ‐ quality reproducible scans of the arterial lumen with the ultrasound probe 5–10 cm proximal to the antecubital fossa, its position documented with a tape measure for accurate repositioning of the probe in the second visit. On the lower arm, a blood pressure cuff was inflated 40 to 50 mmHg above systolic pressure for 5 min to temporarily occlude the brachial artery. According to a fixed ultrasound imaging application protocol, 1 min prior and 3 min after arterial occlusion, electrocardiogram (EKG) ‐ triggered images were recorded in DICOM clips, with each full ‐ frame image in the clip to provide a lumen diameter measurement. For off ‐ line scan analyses, the clips were directly and securely transferred from the ultrasound equipment to the core lab (Imagelabonline & Cardiovascular, Erichem, The Netherlands) for immediate in ‐ study quality control and investigational site feedback by the core lab FMD experts. For the treatment efficacy image analyses, per ‐ subject batches of FMD scans were provided to the trained and certified technicians of the core lab. Technicians were blinded to subject demographic, clinical, and treatment data, as well as scan visit order. Validated and FDA ‐ approved image analysis software was used (Brachial Analyzer, Medical Imaging Applications, Coralville, IA, USA).
FMD was calculated as the percentage of change in the brachial arterial diameter: Brachial FMD ሺ % ሻൌ Maximum diameter ሺ mm ሻെ Baseline diameter ሺ mm ሻ Baseline diameter ሺ mm ሻ ൈ 100
(1)
where the brachial arterial lumen diameter at ‘baseline’ was the average during 1 min prior to cuff ‐ occlusion recording of the brachial artery diameter, and the maximum brachial arterial lumen diameter was the maximum diameter during the 3 min of post ‐ cuff release recording. 2.4. Blood Sampling and Analytical Methods A vein cannula was inserted into the subject’s left arm, and a blood sample (12 mL) was collected shortly after FMD measurement at T0, T1, T2, and T3 during both intervention days. Serum glucose concentrations were measured using the hexokinase method (3L82, ARCHITECT c system, Abbott Laboratories, Abbott Park, IL 60064, USA). Serum insulin levels were determined using a chemiluminescent microparticle immunoassay (8K41, ARCHITECT c system, Abbott Laboratories, USA). Homeostasis model assessment (HOMA) index was calculated using the following equation: fasting glucose (mmol/L) × fasting insulin ( μ U/L)/22.5. 2.5. Statistics Statistical analyses were carried out using SPSS Version 24 and SAS Version 9.3. Sample size was calculated based on primary outcome FMD. On the basis of intra ‐ sonographer repeatability of 1.25%FMD, α of 0.05 (two ‐ sided), and β of 0.1 and on an effect size of 0.9%FMD, the minimal required
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