Acute ingestion of a novel whey-derived peptide improves vascular endothelial responses in healthy individuals: a randomized, placebo controlled trial

Background Whey protein is a potential source of bioactive peptides. Based on findings from in vitro experiments indicating a novel whey derived peptide (NOP-47) increased endothelial nitric oxide synthesis, we tested its effects on vascular function in humans. Methods A randomized, placebo-controlled, crossover study design was used. Healthy men (n = 10) and women (n = 10) (25 ± 5 y, BMI = 24.3 ± 2.3 kg/m2) participated in two vascular testing days each preceded by 2 wk of supplementation with a single dose of 5 g/day of a novel whey-derived peptide (NOP-47) or placebo. There was a 2 wk washout period between trials. After 2 wk of supplementation, vascular function in the forearm and circulating oxidative stress and inflammatory related biomarkers were measured serially for 2 h after ingestion of 5 g of NOP-47 or placebo. Macrovascular and microvascular function were assessed using brachial artery flow mediated dilation (FMD) and venous occlusion strain gauge plethysmography. Results Baseline peak FMD was not different for Placebo (7.7%) and NOP-47 (7.8%). Placebo had no effect on FMD at 30, 60, and 90 min post-ingestion (7.5%, 7.2%, and 7.6%, respectively) whereas NOP-47 significantly improved FMD responses at these respective postprandial time points compared to baseline (8.9%, 9.9%, and 9.0%; P < 0.0001 for time × trial interaction). Baseline reactive hyperemia forearm blood flow was not different for placebo (27.2 ± 7.2%/min) and NOP-47 (27.3 ± 7.6%/min). Hyperemia blood flow measured 120 min post-ingestion (27.2 ± 7.8%/min) was unaffected by placebo whereas NOP-47 significantly increased hyperemia compared to baseline (29.9 ± 7.8%/min; P = 0.008 for time × trial interaction). Plasma myeloperoxidase was increased transiently by both NOP-47 and placebo, but there were no changes in markers inflammation. Plasma total nitrites/nitrates significantly decreased over the 2 hr post-ingestion period and were lower at 120 min after placebo (-25%) compared to NOP-47 (-18%). Conclusion These findings indicate that supplementation with a novel whey-derived peptide in healthy individuals improves vascular function.


Background
The vascular endothelium is a single cell layer lining the lumen of blood vessels that substantially impacts vascular health and disease risk by regulating vasoconstriction and vasodilation, blood pressure, blood clotting, angiogenesis, inflammation, and passage of materials between the circulating blood and the interior components of the vessel wall. An important paracrine factor involved in vascular homoeostasis is endothelium-derived nitric oxide (NO·), which is a potent vasodilator that also inhibits platelet aggregation, inflammatory cell adhesion to the vessel wall, and smooth muscle cell proliferation [1]. Impaired NO· signaling and endothelial dysfunction have been implicated in metabolic syndrome [2] and cardiovascular disease [3]. Therefore, interventions that target NO· and vascular function are relevant for disease prevention.
Numerous therapies targeting the vascular endothelium have been proposed [4]. Nutritional strategies have focused on antioxidants (e.g., vitamin E, vitamin C, polyphenols, etc.) because oxidative stress contributes to endothelial dysfunction. Antioxidant supplementation mitigates postprandial endothelial dysfunction associated with high carbohydrate and high fat meals [5]. Oral supplementation [6] and intra-arterial administration [7] of L-arginine, the rate limiting amino acid for endothelial NO· synthesis [8], restored impaired endothelial function in older individuals and in response to a high fat meal [9], but not in younger adults with normal endothelial function [10].
Bioactive peptides derived from food, especially milk proteins, have been shown to exert a wide range of biological actions including decreased blood pressure [11,12] and improved endothelial function [13]. Milk is a rich source of angiotensin-converting enzyme (ACE) inhibitory peptides [14]. ACE inhibition prevents the conversion of angiotensin I to angiotensin II, a potent vasoconstrictor. Several clinical studies have shown improvement in endothelial function in patients prescribed ACE inhibitors [4], which could be the result of pleiotropic effects of ACE inhibitors on the vascular endothelium [15].
A search for isolates from whey protein hydrolysates that could increase NO production was carried out by Glanbia Nutritionals. A nitric oxide peptide (NOP-47) was identified that was shown to increase NO synthesis in vitro as determined by the analysis of NO· metabolites in human pulmonary artery endothelial cells (HPAE-26) (data provided by Glanbia Nutritionals). To extend upon these findings, we conducted a randomized, double-blind, cross-over trial to determine if NOP-47 affected vascular physiology in healthy human volunteers. We hypothesized that a single dose of NOP-47 would enhance vascu-lar function as measured by flow-mediated dilation (FMD) of the brachial artery using high-frequency ultrasound [16] and reactive hyperemia forearm blood flow assessed by venous occlusion plethysmography [17]. Forearm FMD measures dilation in a conduit artery and is considered an index of NO· bioavailability [18] that is also correlated with coronary artery endothelial function [19] and cardiovascular disease risk and mortality [20]. Reactive hyperemia venous occlusion plethysmography measures vasodilation in the resistance vessels, and is not appreciably affected by NO· in human forearms [21]. A secondary objective was to characterize the effects of NOP-47 on circulating markers of antioxidant capacity, oxidative stress, and inflammation since these factors have been demonstrated to influence vascular function though various biologic mechanisms.

Study design
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.

Subjects
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.

Supplementation protocol
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.

Testing protocol
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 Study timeline (A) and vascular testing protocol (B)  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.

Flow mediated dilation
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 occlusion [16]. 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 Values are means ± SD. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate. 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.

Strain gauge plethysmography
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.

Blood collection and biochemical analyses
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 (Car-dioMPO, 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 (NO 2 -/NO 3 -) 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 cutoff 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).

Statistical analyses
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 productmoment correlation coefficient. The α-level for significance was set at 0.05.

Results
Body mass remained stable over the course of the study (mean ± SD: 70.01 ± 9.27 kg versus 69.59 ± 8.85 kg for visits 1 and 4, respectively). There were also no significant changes in systolic or diastolic blood pressure after 2 wk of supplementation with NOP-47 and placebo. There were no adverse responses reported by subjects during either trial. All but one subject correctly identified which supplement contained NOP-47.
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.

Hematological responses
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.  (Table 3).

Discussion
We tested the effects of a novel peptide derived from whey on vascular endothelial function in healthy, young men and women. Peripheral vascular function was assessed in a conduit vessel using FMD of the brachial artery by highresolution ultrasound and in forearm resistance vessels using venous occlusion plethysmography. We demonstrated that 2 wk of supplementation had no effect on fasting measures of vascular function, but acute ingestion of NOP-47 significantly increased postprandial FMD at 30, 60 and 90 min post-ingestion and reactive hyperemia forearm blood flow measured at 120 min post-ingestion. Shear stress induced dilation of conduit vessels like the brachial artery are principally regulated by the potent vasodilator NO· [28], whereas dilation of resistance vessels in response to reactive hyperemia is largely independent of NO· [21]. Therefore acute ingestion of NOP-47 likely enhanced vascular endothelial function through mechanisms that were dependent as well as independent of NO·.
To define the NOP-47 mediated activity on vasodilation, we measured NO· status by evaluating total nitrite and nitrate levels (NO x ), the final metabolites of NO·. We detected a time-dependent decrease in plasma NO x during the 2 h testing period that was partly inhibited after ingestion of NOP-47 at 120 min ( Figure 4). Previous studies have shown a decline in postprandial NO x and decreased FMD after both high fat and high carbohydrate meals [29]. Lower NOx levels correlate with reduced FMD in patients with endothelial dysfunction [30] and in healthy young men and women [31]. Whether better maintenance of NO· after NOP-47 ingestion contributed to the enhanced vascular responses in this study remains unclear.
Although NO x correlates with FMD, the assay is not specific for endothelial NO· production and could also reflect NO· derived from neuronal and inducible NO· synthase [32]. Alternatively, other factors besides NO· may be responsible for the enhanced dilation. Alterations in the balance between vasodilators (e.g., bradykinin, adenosine, vascular endothelial growth factor, and prostacyclin) and vasoconstrictors (e.g., endothelin, prostanoids, and angiotensin II) have been suggested to contribute to the FMD response [33]. Experiments that involve infusion of N G -nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis, would help to elucidate whether the enhanced FMD response to NOP-47 is NO·-dependent.
Whey derived peptides showing ACE inhibitory effects are released during normal digestion in the gastrointestinal tract by proteases. Commonly used enzymatic procedures in the manufacturing of whey hydrolysates also result in rich sources of ACE inhibitory peptides. In order for oral ingestion of whey peptides to exert hypotensive or other biological effects in vivo, it must be absorbed intact and be transported to the target tissue while escaping destruction from intestinal brush border or serum peptidases. Evidence exists to support that peptides are absorbed intact   Plasma total nitrites/nitrates (NOx), normalized to baseline, responses to ingestion of a whey peptide (NOP-47) or Pla-cebo Figure 4 Plasma total nitrites/nitrates (NOx), normalized to baseline, responses to ingestion of a whey peptide (NOP-47) or Placebo. There was a significant main time effect. Significant differences between NOP-47 and Placebo (*P < 0.05).  [34]. Indeed, a specific whey-derived heptapeptide having ACE inhibitory activity was demonstrated to be bioavailable [35].
A possible mechanism by which whey peptides might improve endothelial function is through ACE inhibition. Human clinical trials have shown improvement in endothelial function in patients taking ACE inhibitors (reviewed in [4]), which could be the result of pleiotropic effects of ACE inhibitors on the vascular endothelium [15]. A further mechanism by which whey peptides could affect vascular function is by increasing arginine availability, the rate limiting substrate for nitric oxide synthesis. However, NOP-47 contained approximately 135 mg of arginine, well below the doses previously demonstrated to improve FMD [36].
Most nutraceutical interventions that reported favorably effects on FMD were studied after the ingestion of meals that induced oxidative and inflammatory stress to the endothelium leading to vascular dysfunction [5]. In this study, our primary objective was to examine the effects of NOP-47 in the fasted state without the confounding effects of other nutrients in healthy non-hypertensive individuals with presumed normal endothelial function-ing. There was however a significant increase of ~15% in plasma MPO one hour after ingestion of both NOP-47 and placebo suggesting the protocol induced a small transient elevation in neutrophil activation and oxidative stress. The ischemia caused by repeated forearm arterial and venous occlusions performed during the vascular function tests may have produced brief periods of turbulent flow causing an increase in MPO [37,38]. The increase in MPO at 60 and 90 min after ingestion of both NOP-47 and placebo coincided in time with the significant decrease in NOx. Elevated MPO levels have been shown to interfere with endothelial NO· action and are highly associated with impaired FMD [39].