This study was approved by the Institutional Review Board for use of human subjects in research at the University of Connecticut. All subjects provided written informed consent after having the risks of the study carefully explained to them. A randomized, placebo-controlled, crossover study design with a washout period was conducted. Subjects participated in two vascular testing days with each preceded by 2 wk of daily supplementation with either a whey-derived peptide (NOP-47) or placebo. The order of supplementation was balanced. Following the completion of the first 2 wk supplementation period and first day of vascular testing, participants underwent a minimum 1 wk washout period after which they started the second 2 wk supplementation period consuming the alternative supplement. Each subject reported to the lab for four separate visits (Figure 1, top). In order to eliminate confounding influences on the experimental variables subjects were instructed to fast for 12 h, avoid alcohol, caffeine, and exercise for 24 h, and to consume 1 L of water the night before the visit and 480 mL the morning of the visit to ensure adequately hydration.

Healthy volunteers (n = 20) between 21–39 y were studied (Table 1). Exclusion criteria for subjects included overt chronic diseases as determined by medical history questionnaire, hypertension, smoking, use of vasoactive medications or supplements, or weight change greater than 2.3 kg in the past 3 mo. Women were screened to determine menstrual history and were excluded if hormonal contraceptive use was initiated or changed within the past 3 mo.

The active supplement was a proprietary peptide isolated from a whey protein hydrolysate (NOP-47, Glanbia Nutritionals, Twin Falls, ID) (Table 2). A daily dose of 5 g was pre-measured and placed in individual packets with artificial sweetener. The placebo was identical except the packets contained only artificial sweetener (aspartame and acesulfame potassium). Subjects were provided a 2 wk supply and instructed to consume one packet per day mixed in 300 mL water. Compliance was 100% as assessed by written documentation in log books and verified by study personnel. On the morning of vascular testing, subjects consumed one packet containing 5 g of NOP-47 or placebo mixed in water in the presence of an investigator not directly involved in vascular data collection nor analysis at the completion of the study. Subjects ingested the beverage within 3 min, after which a timer was started for the 2 h postprandial testing protocol. A questionnaire to address subjective symptoms and side effects associated with each supplement was administered at the end of the study.

Upon arrival to the laboratory, participants provided a urine sample and hydration state was confirmed by measurement of urine specific gravity (USG) with a handheld refractometer. A USG < 1.020 indicated euhydration. If USG was > 1.020, then participants were instructed to drink water until their USG was < 1.020. Body mass was measured to the nearest 0.1 kg on a calibrated digital scale.

Visits 1 and 3 consisted of anthropometric measurements, detailed instructions on filling out dietary records, distribution of supplements, and a single venous blood draw obtained in a supine position. Visits 2 and 4 occurred on two occasions at the same time of the day after 2 wk supplementation with NOP-47 or placebo. A flexible catheter was inserted into a left forearm vein and after a 15 min supine stabilization period, blood samples were collected from a 3-way stopcock connected to the end of the catheter for fasting baseline and subsequent postprandial biochemistry measurements. Next fasting measurements of FMD and forearm blood flow (FBF) (described below) were determined. Fifteen minutes of recovery were allowed between FMD and FBF measurements before ingestion of the test beverage. Following these baseline measurements, subjects consumed a single 5 g dose of either NOP-47 or placebo mixed in 300 mL of water and with artificial sweetener. Post-ingestion FMD and FBF measurements were made intermittently (Figure 1, bottom). Blood samples were obtained at 15, 30, 45, 60, 90, and 120 min post-ingestion. Subjects remained supine in a comfortable position for the entire duration of the test. To ensure standardization between testing trials subjects were instructed to maintain their current level of physical activity during the study period and to replicate their dietary intake from previously recorded diet records the day prior to each vascular testing visit. Women were assessed during the same phase of their individual menstrual cycle as to account for any changes in vascular function 22 or blood markers due to menstrual phase.

FMD was assessed using standardized procedures for performing high-frequency ultrasonographic imaging before (PRE) and at 30, 60, and 90 min after ingestion of the test beverage. The technique provokes the release of NO·, resulting in vasodilation that can be quantitated as an index of vasomotor function 16. All tests were performed in a quiet, temperature-controlled room after a 10 min period in a supine position. A blood pressure cuff was placed on the upper right arm for occlusion. ECG leads were attached to monitor heart rate throughout the procedure. The brachial artery was imaged above the antecubital crease, and the transducer was placed to image the brachial artery in a longitudinal axis with clear visualization of the anterior and posterior vessel walls. When a clear image of the anterior and posterior walls of the artery was obtained, the transducer was held by a stereotactic clamp and the position held constant for the duration of the data collection. After optimization of the image, baseline brachial artery diameter was recorded for 30 heart beats. A mark was made on the arm where the image was collected. The cuff was inflated to 200 mm Hg for 5 min using a rapid cuff inflator (Hokanson E20, Bellevue, WA, USA) to occlude the brachial artery, and then released. Arterial diameter was then assessed continuously for 300 heart beats after occlusion16. Images of the brachial artery were obtained using an Acuson 13.0-MHz linear array transducer and an Aspen cardiac ultrasound system (Acuson Corp, Elmwood Park, NJ). Anatomical measurements were made to ensure placement of the transducer in the same location on the arm during the second visit. Image analysis was performed using MIA software (Medical Imaging Applications, Iowa City, IA, USA). For baseline, the average diameter taken from 30 frames was used. Three hundred frames were recorded from the postocclusion period. Peak postocclusion diameter was calculated by averaging the vessel diameter 5 frames immediately before the observed peak diameter and the 5 frames immediately after the same mark. Brachial artery FMD was calculated and expressed as a percentage of the baseline diameter 23. All vascular measurements and analysis were performed by the same person. Using the same investigative team, coefficients of variation for arterial diameter on repeat scans with repositioning on a group of men and women (N = 10) in our laboratory were 2.2% for measurements made the same day, and 2.2% for measurements made on two consecutive days.

Forearm blood flow was measured from the same arm as FMD using venous occlusion strain gauge plethysmography. A calibrated indium-gallium filled silastic strain gauge, encircled around the largest diameter of the right forearm, was connected to a plethysmograph (EC6, Hokanson, Inc., Bellevue, WA, USA). The increase in forearm volume was measured after blocking the venous efflux by an upper arm cuff inflated to 50 mmHg by a rapid cuff inflator (Hokanson E20, Bellevue, WA, USA) for 7 sec during each 15 sec cycle to determine resting forearm blood flow (R-FBF). This measurement was performed at rest (PRE) and in between the FMD protocol at 20, 50, 80, and 110 min after ingestion of the test beverage. The hand circulation was excluded by a wrist cuff inflated to 220 mmHg for 1 min before and during each flow evaluation. The forearm blood flow was estimated using specialized software (Noninvasive Vascular Program 3 (NIVP3), Hokanson, Bellevue, WA, USA) which calculated the slope from the change in forearm volume over time and determined blood flow as percent volume change per minute (%/min). Four plethysmographic measurements were averaged to obtain values for R-FBF. To determine reactive hyperemia induced forearm blood flow (RH-FBF), a blood pressure cuff on the upper right arm was inflated to a pressure of 200 mmHg for 5 min. FBF was determined as described above upon release of the occlusion. This measurement was performed at rest (PRE) and 120 min after ingestion of the test beverage.

All blood samples were obtained from the left arm vein while participants rested quietly in the supine position. Whole blood was collected into tubes with no preservative or lithium heparin and centrifuged (1500 × g, 15 min, 4°C). Serum/plasma was transferred into storage tubes, snap frozen in liquid nitrogen, and stored at -80°C for future analysis. Samples for each asasy were analyzed in duplicate. Myeloperoxidase was measured from lithium heparin plasma by high-sensitivity sandwich ELISA (CardioMPO, Prognostix, Cleveland, OH) (CV = 5.9%). Serum glucose concentrations were analyzed using a YSI glucose/lactate analyzer (YSI 2300 STAT, Yellow Springs, OH). Total nitrite/nitrate (NO₂^-/NO₃^-) was measured as an estimate of NO· production using a colorimetric kit (Cayman Chemical, Ann Arbor, MI, USA) in accordance with the manufacturer's instructions (CV = 8.0%). Serum samples were filtered using 10 kDa molecular weight cut-off filters prior to analysis to reduce background absorbance. Plasma C-reactive protein (CRP) was determined on an IMMULITE Automated Analyzer using the commercially available immulite chemiluminescent enzyme immunometric assay (Immulite^®, Diagnostic Products Corp., Los Angeles, CA, USA).

Total plasma antioxidant status was determined using the ferritin-reducing ability of plasma (FRAP) assay as previously described 2425. Briefly, diluted plasma (1:4) was mixed on a 96 well plate with 300 μl of freshly prepared and pre-warmed (37°C) FRAP reagent [50 ml of sodium acetate buffer (300 mmol/L), 5 ml of TPTZ reagent prepared in 40 mmol/L HCl, and 5 ml of FeCl₃ (20 mmol/L)]. Following incubation (15 min, 37°C), samples were read at 593 nm on a microplate reader (SpectraMax M2, Molecular Devices Corporation, Sunnyvale, California, USA) and FRAP concentrations were calculated using trolox standards that were prepared in parallel (CV = 4.3%).

Plasma malondialdehyde (MDA) was measured by HPLC-FL as described previously 26 with minor modifications. In brief, 200 uL of plasma was mixed with 150 uL of 5% (w/v) TCA. After centrifugation (4,000 × g, 10 min, 4°C), the supernatant was thoroughly mixed with 50 μL of 0.6% (w/v) thiobarbituric acid. The sample was incubated (1 h, 100°C), then rapidly chilled in an ice bath, followed by the addition of 225 μL methanol and 25 μL 1 N NaOH. The supernatant was collected following centrifugation (16,000 × g, 4°C, 10 min) and subsequently injected (20 μl) on the HPLC system (Beckman Coulter; Fullerton, CA). The sample was separated on a C₁₈ separation column (250 × 4.6 mm i.d.; 5 μm; Phenomenex; Torrance, CA) under isocratic (0.9 ml/min) conditions using 60:40 methanol and 50 mM phosphate buffer (pH 5.5) as the mobile phase. MDA was detected using excitation and emission settings of 532 nm and 553 nm, respectively, and was quantified against MDA standards that were prepared in parallel from 1,1,3,3-tetramethoxypropane.

Serum cytokines and chemokines [tumor necrosis factor-alpha (TNFα), interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), soluble E-selectin (sE-Selectin), soluble vascular cell adhesion molecule-1 (sVCAM-1), and soluble intracellular adhesion molecule-1 (sICAM-1)] were measured using xMAP^® technology on a Luminex^® IS 200 system with antibodies to these analytes from LINCO Research (St. Charles, MO)27. Assays were completed according to manufacturer's instructions.

Forearm FMD data was analyzed with a 2 × 4 ANOVA with supplement trial (NOP-47 vs Placebo) and time (pre, 30, 60, and 90 min) as within effects. Sex was also included as an effect, but not found to be statistically significant and thus men and women were combined in all analyses. Significant main or interaction effects were further analyzed using a Fishers LSD post hoc test. Relationships among selected variables were examined using Pearson's product-moment correlation coefficient. The α-level for significance was set at 0.05.

Pre-occlusion diameters were not significantly different before ingestion of the NOP-47 (3.91 mm) and placebo (3.90 mm) and remained remarkably stable over time during both trials (range 3.89 to 3.91 mm) indicating maintenance in vascular tone over the postprandial period as well as a high degree of reproducibility in probe placement and measurement of the artery. Peak FMD (mean; 95% CI) significantly increased at 30 (8.87; 7.28–10.46%), 60 (9.94; 7.94–11.94%), and 90 (9.02; 7.41–10.63%) min post-ingestion in the NOP-47 trial which were significantly higher than corresponding placebo time points at 30 (7.52; 5.90–9.07%), 60 (7.21; 5.76–8.65%), and 90 (7.61; 5.91–9.31%) min (P < 0.0001 for time × trial interaction) (Figure 2, top). Individual responses revealed that 15 out of 20 subjects had greater peak FMD at 60 min (Figure 2, bottom) and 90 min post-NOP-47 ingestion compared to these same time points following ingestion of placebo.

Reactive hyperemia forearm blood flow was assessed in response to 5 min of cuff occlusion by venous occlusion plethysmography before and 120 min after supplement ingestion. Maximal hyperemic blood flow was similar after 2 weeks of supplementation with NOP-47 (27.6 ± 7.6%/min) and placebo (27.6 ± 7.2%/min). The response to acute ingestion showed a significant increase at 120 min for NOP-47 (29.9 ± 7.5; 95% CI = 26.25–33.58%/min) and no change for placebo (27.5 ± 7.8; 95% CI = 23.51–30.84%/min) (P = 0.008 for time × trial interaction) (Figure 3). Resting forearm blood flow was also assessed before supplement ingestion and 20, 50, 80, and 110 min post-ingestion. Compared to hyperemic blood flow, resting blood flow values were considerably smaller in magnitude and only showed a significant main time effect (P = 0.002) as reflected by a significant increase at 110 min compared to pre-ingestion.

Hematoligical responses are presented in Table 3. Serum glucose was unaffected by 2 wk of supplementation or acute ingestion. Plasma nitrites/nitrates (NO_x) decreased significantly over time (P < 0.001; Figure 4) and specific post-hoc effects were observed at 90 and 120 min compared to pre-ingestion. There was a greater decline in subjects consuming the placebo at the 120 min time point (P = 0.03). There was a significant time effect for serum FRAP (P = 0.001) with values at 30 min higher than baseline. Fasting plasma MPO was not affected by 2 wk of supplementation. However, there was a significant main time effect in response to acute supplementation ingestion with values increasing significantly at 60 and 90 min (P = 0.003 for time effect). Plasma CRP, MDA and several inflammatory markers were unaffected by chronic or acute ingestion (Table 3).