Parameters reflecting cardiovascular health and inflammation were studied in a pilot clinical trial conducted on 40 patients with prehypertension. The patients were treated with a new proprietary formulation of a whey protein (WP) isolate embedded into lycopene micelles (WPL) during a 1-month period. Control groups received lycopene or WP as a singular formulation or placebo pills for the same period of time. Combined WPL formulation of whey protein and lycopene has caused multiple favorable changes in the cardiovascular function (including a tendency to the reduced systemic blood pressure), the plasma lipid profile, and the inflammatory status of patients with prehypertension, whereas singular formulations of the compounds and placebo did not have such an effect. The reduction of plasma triglycerides and cholesterol fractions and almost two-fold decline in C-reactive protein (CRP) and inflammatory oxidative damage (IOD) levels as well as an increase in nitric oxide (NO), tissue oxygenation (StO2), and flow-mediated dilation values constitute the most significant benefit/outcome of the treatment with the combined formulation of whey protein and lycopene. The treatment did not affect the values of ankle-brachial index (ABI), body weight, and body mass index (BMI).
Whey proteins and whey-related peptides represent a newly emerging class of biological substances with as yet poorly understood potential benefits for human health. For decades whey has been considered as a fairly useless liquid byproduct from the production of cheese. However, modern methods for the fractionation of whey allow the isolation of a variety of substances with an enormously wide spectrum of biological activity [
Moreover, there is a growing number of whey-derived peptides whose pharmacological activity
Despite their high inhibitory potency, whey-derived proteins and peptides are highly susceptible to gastrointestinal enzymes which undermine their oral use in treatment of hypertension. This is the main reason why most clinical trials on whey proteins or their derivatives require a significant quantity to be consumed daily. Indeed, susceptibility of whey protein and its derivates to the intestinal enzymes is a major problem complicating their therapeutic use. It explains variations in individual responses to whey protein administration among patients and dictates the necessity for the development of new whey protein formulations development providing a degree of protection for the whey protein and its peptides from the intestinal environment. This would consequently increase their bioavailability and, as result, of this their efficacy.
In the present paper, we report the results of a pilot clinical trial with a new proprietary formulation of a whey protein (WP) isolate embedded into lycopene micelles [
The study was conducted at the Institute of Cardiology, the Ministry of Health of the Russian Federation (Saratov, RF), during 2010-2011. The protocol was approved by the local ethics committee. All patients were informed about the purpose of the study and have given written consent regarding their participation in the study.
The study was part of a larger multiarm trial for which 107 patients with prehypertension fulfilling clinical inclusion criteria were selected. Prehypertension was defined according to the USA Joint National Committee, JNC 7 [
Caucasian male or female subjects 45–73 years old. Signed informed consent. Non- or light-to-moderate smokers (≤10 cigarettes daily). Sustained resting systolic blood pressure 120–139 mm Hg. Sustained resting diastolic blood pressure 80–89 mm Hg. Serum markers for inflammation Chl.pn-IgG ELISA × 103 ≥ 400 and CRP ≥ 6 Serum markers for oxidative stress LDL-Px ELISA × 103 ≥ 250 and IOD ≥ 40 No antihypertensive, lipid-lowering or any other cardiovascular drugs. Willingness and ability to comply with the protocol for the duration of the study.
Unwillingness to sign informed consent. Unable to comply with the protocol for the duration of the study. History of MI in the 3 months preceding the study. Ejection fraction (EF) < 45%. Significant medical condition that would impact safety considerations (e.g., significantly elevated LFT, hepatitis, severe dermatitis, uncontrolled diabetes, cancer, severe GI disease, fibromyalgia, renal failure, recent CVA (cerebrovascular accident), pancreatitis, respiratory diseases, epilepsy, etc.). Compulsive alcohol abuse (>10 drinks weekly), or regular exposure to other substances of abuse. Participation in other nutritional or pharmaceutical studies. Resting heart rate of >100 beats per minute or <45 beats per minute. Positive test for tuberculosis, HIV, or hepatitis B. Did not tolerate phlebotomy. On a special diet in the 4 weeks prior to the study (e.g., liquid, protein, raw food diet). Tomato or milk intolerance.
The first product was whey protein isolate (WP), Prolacta (Lactalis, France). The daily dose was given as 1 capsule containing 70 mg of WP. The second control product was lycopene (Vitatene, Spain). The daily dose was 1 capsule containing 7 mg of lycopene. The third product was a complex of WP embedded into protective lycopene matrix, whey protein lycosome (WPL, Lycotec, UK) [
To measure body mass index, BMI, body mass of the patients, and their height were measured in the morning and BMI was calculated in kg/m2. Pulse rate, systolic and diastolic blood pressure, SBP, and DBP were measured three times in the left arm of the seated patient after 15 min of rest. The time between measurements was no less than 2 minutes. The mean number for each parameter was calculated.
All body and vascular parameters were measured in the morning between 8 and 10 am.
Endothelium-dependent flow mediated vasodilatation was measured in accordance with traditional guidelines accepted in the last 10 years [
High resolution ultrasound was used at the same anatomical landmark of a section of the brachial artery for a period of 30 sec before and during the peak of reactive hyperaemia. It was applied prior to sphygmomanometer cuff occlusion and 1 min after its deflation. The level of inflation was 50 mm Hg above the patient’s systolic blood pressure and it lasted for 5 minutes. Arterial diameter was imaged above the antecubital fossa in a longitudinal scan by duplex ultrasound with linear phase-array transducer. FMD was calculated as a change in poststimulus diameter as a percentage of the baseline diameter [
Between left and right brachial arteries, the one with the highest SBP, and between left and right tibial arteries, the one with the highest SBP were chosen for the assessment of ABI. For this purpose, a continuous-wave Doppler probe was used after patients had been in a supine position for at least 15 min of rest [
As a tissue target for the assessment of oxygen saturation, StO2, or combined level of oxygenated haemoglobin and myoglobin, we used the the Nar eminence and forearm muscles of the patients. StO2 was analysed by continuous wavelength near-infrared spectroscopy, NIRS, with wide-gap second-derivative (In Spectra, Hutchinson Technology, MN, USA). The measurements were made at different time points. The recording was started after 15 min of rest in a supine position before occlusion of the brachial artery. It was then continued during stagnant ischemia induced by rapidly inflating the cuff to 50 mm Hg above systolic BP. The ischemia lasted for 3 min, and the recording period lasted for another 5 min after that until StO2 was stabilized [
Then the area under the hyperaemic curve, AUC, of the recorded signal for the settling time in the postocclusion period was calculated as described earlier in % O2/minute [
Blood was collected in the morning after night fast from arm veins of the patients. The plasma was separated from the rest of the clotted mass by centrifugation, then aliquots were stored at −80°C prior to analysis.
Glucose, total cholesterol, TC, triglycerides, TG, high density cholesterol, HDL, low density cholesterol, LDL, C-reactive protein, CRP,
Plasma samples were incubated overnight in 0.05 M PBS acetate buffer (pH 5.6) which would imitate the type of oxidative damage which occurs during the release of lycosomes following neutrophil degranulation. The following morning, the reaction was terminated using trichloroacetic acid. The concentration of the end products such as malonic dialdehyde (MDA), and other possible thiobarbituric acid reactive substances, TBARS, was then measured by colorimetric methods [
Activity of serum LDL peroxidase proteins, which include IgG with superoxide dismutase activity [
For the assessment of normally distributed parameters, the Shapiro-Wilk method was used. Student’s
Table
Clinical groups.
Parameters | Patient group |
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WP | Lycopene | WPL | Placebo |
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Number of patients | 10 | 10 | 10 | 10 |
Male/female | 6/4 | 4/6 | 5/5 | 5/5 |
Age | 57.8 ± 3.5 | 52.1 ± 5.6 | 58.4 ± 3.2 | 51.1 ± 5.2 |
Light or moderate smokers | 2 | 1 | 2 | 2 |
Body mass index, in kg/m2 | 25.9 ± 2.8 | 28.3 ± 3.1 | 27.2 ± 3.4 | 26.8 ± 5.7 |
Fasting glucose, mg/dL |
85 ± 4.5 |
82 ± 3.9 |
67 ± 6.8 |
79 ± 5.2 |
As can be seen from Table
Changes in lipid profile in prehypertensive patients treated with whey protein and lycopene formulations.
Product | TC | TG | LDL | HDL |
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in mg/dL | in mg/dL | in mg/dL | in mg/dL | |
WP baseline after 1 month ( |
215 ± 9.9 | 147 ± 10.1 | 133 ± 13.2 | 40.7 ± 0.6 |
222 ± 11.3 | 151 ± 12.3 | 132 ± 11.5 | 40.9 ± 0.7 | |
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Lycopene baseline after 1 month ( |
199 ± 10.2 | 145 ± 9.4 | 142 ± 9.9 | 38.5 ± 0.7 |
191 ± 9.8 | 139 ± 10.1 | 141 ± 8.7 | 39.1 ± 0.6 | |
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WPL baseline after 1 month ( |
209 ± 9.1 | 148 ± 14.5 | 159 ± 11.0 | 39.6 ± 1.2 |
180 ± 8.0 | 97.5 ± 7.5 | 132 ± 7.8 | 44.5 ± 1.1 | |
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Placebo baseline after 1 month ( |
185 ± 10.3 | 135 ± 11.2 | 146 ± 12.4 | 40.1 ± 1.0 |
182 ± 9.6 | 141 ± 10.5 | 144 ± 11.7 | 40.6 ± 0.9 | |
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As shown in Table
Effect of markers of oxidative stress, inflammation, and NO.
Product | Chl.pn-IgG | CRP | LDL-Px | IOD |
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ELISA × 103 | in |
ELISA × 103 | in |
in |
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WP baseline after 1 month ( |
639 ± 55 | 8.0 ± 1.0 | 482 ± 53 | 97 ± 11 | 31 ± 6.3 |
664 ± 62 | 7.4 ± 0.6 | 467 ± 62 | 94 ± 10 | 29 ± 5.5 | |
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Lycopene baseline after 1 month ( |
842 ± 85 | 12.1 ± 1.7 | 461 ± 44 | 93 ± 11 | 26 ± 4.8 |
754 ± 61 | 10.3 ± 1.6 | 413 ± 42 | 67 ± 9 | 30 ± 5.1 | |
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WPL baseline after 1 month ( |
725 ± 63 | 15.3 ± 2.2 | 410 ± 61 | 88 ± 9 | 23 ± 5.6 |
342 ± 27 | 7.8 ± 1.5 | 295 ± 45 | 48 ± 5 | 38 ± 5.2 | |
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Placebo baseline after 1 month ( |
720 ± 81 | 7.2 ± 3.1 | 470 ± 43 | 86 ± 10 | 25 ± 4.4 |
759 ± 79 | 7.4 ± 3.9 | 495 ± 61 | 85 ± 12 | 27 ± 4.1 | |
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None of the treatments affected values for pulse rate and ABI (Table
Effect on cardiovascular parameters.
Product | Pulse rate per min | ABI |
FMD (%) | Blood pressure, in mm Hg | StO2, % O2/min | |
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Systolic | Diastolic | |||||
WP baseline after 1 month ( |
71.0 ± 3.9 | 1.05 ± 0.04 | 9.4 ± 1.1 | 137 ± 9.7 | 86.1 ± 5.0 | 10.2 ± 0.9 |
70.6 ± 2.8 | 1.06 ± 0.03 |
9.5 ± 1.2 | 135 ± 8.8 | 83.6 ± 3.2 | 10.3 ± 1.1 | |
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Lycopene baseline after 1 month ( |
73.1 ± 3.8 | 1.09 ± 0.05 | 10.5 ± 1.1 | 132 ± 3.6 | 86 ± 3.4 | 13.1 ± 0.8 |
71.5 ± 4.9 | 1.01 ± 0.07 |
11.1 ± 0.9 | 130 ± 6.5 | 83 ± 4.8 | 14.7 ± 0.9 | |
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WPL baseline after 1 month ( |
68.3 ± 3.2 | 0.97 ± 0.07 | 10.3 ± 0.8 | 131 ± 8.0 | 81 ± 5.0 | 11.4 ± 0.6 |
67.9 ± 2.9 | 0.97 ± 0.08 | 12.9 ± 0.8 | 124 ± 7.4 | 77 ± 4.1 | 16.4 ± 0.8 | |
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Placebo baseline after 1 month ( |
65.2 ± 5.1 | 0.97 ± 0.07 | 10.2 ± 0.9 | 135 ± 8.5 | 84 ± 4.1 | 11.5 ± 0.7 |
66.4 ± 4.6 | 0.97 ± 0.08 | 10.0 ± 1.2 | 136 ± 9.6 | 83.5 ± 3.3 | 12.2 ± 0.4 | |
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Prehypertension is a progressive human disease which often develops without any specific identifiable cause and involves multiple functional changes in microcirculation, inflammatory response, and plasma lipid profile [
The major finding of the clinical trial reported above is that a combined WPL formulation of whey protein and lycopene causes multiple favorable changes in cardiovascular function (including tendency to the reduction of the systemic blood pressure), plasma lipid profile, and inflammatory status of patients with prehypertension, whereas singular formulations of the compounds do not have such an effect. The reduction of plasma triglycerides and cholesterol fractions and almost two-fold decline in CRP and IOD levels as well as an increase in
These changes in inflammatory activity were accompanied by a reduction in the level of oxidative stress. We cannot claim that they were the consequence of anti-inflammatory activity of WPL or that the whey protein that isolates itself contained antioxidant components which were able to take effect when they were delivered in the lycosome format.
Level of NO production, flow mediated dilation, and tissue oxygenation are inversely correlated to markers of inflammation and oxidative stress. Therefore, we again cannot be certain here whether the improvement of these parameters is a result of anti-inflammatory and anti-oxidant activity of WPL or a result of intrinsic vasoactive molecules in the protein isolates itself. Or perhaps it was a combination of both possibilities.
In conclusion, it is possible to say that the improved protection of whey protein product by lycosome could allow people to improve their metabolic parameters, vascular function, and antiageing microcirculation. Perhaps this resembles the similar practice of embedding cheese and whey products into lycopene-rich tomato sauce which is one of the factors behind the longevity and cardiovascular health provided by the Mediterranean diet.
Although additional studies are required to confirm and extend our results, the results presented above represent a significant step forward in the management of hypertension and cardiovascular disease.
The authors declare that they have no conflict of interests.
This work is supported by Lycotec Ltd., UK.