IPP-rich milk protein hydrolysate lowers blood pressure in subjects with stage 1 hypertension, a randomized controlled trial

Male and female Caucasian subjects were recruited from the pool of volunteers of TNO Quality of Life. After being informed about the study, the subjects gave voluntary written informed consent. Health was assessed by an interview on medical history, physical examination, and routine laboratory tests on blood and urine. To select subjects with prehypertension and stage 1 hypertension, the classification according to JNC-7 was used [24]: subjects with a systolic BP between 140 - 159 mm Hg or a diastolic BP between 90 - 99 mm Hg were assigned to 'stage 1 hypertension', and subjects with a systolic BP between 120 - 139 mm Hg or a diastolic BP between 80 - 89 mm Hg were designated 'prehypertension'. BP was measured on three separate visits. Subjects with a history of medical events or medication that may have influenced the study outcome were excluded from participation. Subjects who were on slimming, medically prescribed, vegan, vegetarian or macrobiotic diet were also excluded as were smokers and subjects using more than 28 units (men) or 21 units (women) of alcohol per week to exclude alcoholics. Pregnant or lactating women were also excluded from participation. Eighty healthy subjects were included in the study.

The study was designed as a randomized, placebo-controlled, double blind, crossover study. Study treatments were given to the subjects according to a Latin square design. In view of the very short half-life of lactotripeptides [25], no wash-out period was included between treatments. The study was powered to detect a 3.5 mmHg systolic blood pressure difference between placebo and treatment at an α of 0.05 (two-sided) and a β of 0.8.

Subjects visited the test facility of TNO Quality of Life at three consecutive test days at the end of each treatment period. At the first two test days, BP measurements were performed two hours after the subjects had their own habitual breakfast and the study substances at home. At the third test day, subjects came in a fasted state for collection of blood and spot-urine samples. No alcohol and sports were allowed the day before the test days, and no dairy products were allowed at breakfast on the test days. During the study period, subjects were instructed not to consume more than one portion of a fermented dairy product per day.

The study was performed according to the ICH Guideline for Good Clinical Practice (ICH topic E6, adopted 01-05-1996 and implemented 17-01-1997) and was approved by the independent Medical Ethics Committee METOPP (Tilburg, the Netherlands).

MPH1 and MPH2 were produced through hydrolysis of glycomacropeptide and casein, respectively. Hydrolysis was performed using a proline-specific endoprotease. The nutritional properties, as well as the amounts of bioactive peptides of the study products are presented in table 1. Lactotripeptides were quantified as described in [26].

Study treatments were offered to the volunteers in capsules, to exclude any pre-ingestion interference with food matrices. The treatments consisted of consumption of MPH1 or MPH2 or placebo during 4 weeks. Five hundred mg hydrolysed protein was used per capsule for each of the MPH treatments. The lactotripeptide content per capsule was 7.5 mg IPP for MPH1, and 6.6 mg MAP, 2.3 mg LPP, and 1.8 mg IPP for MPH2. Subjects received 2 capsules per day, and were instructed to consume one capsule directly upon completion of breakfast and one capsule directly upon completion of dinner. Placebo treatment consisted of daily consumption of 2 capsules, each of them filled with 500 mg cellulose (food grade quality).

BP was measured between 2.5 and 3.5 h after ingestion of the study substances using automated digital sphygmomanometry (OMRON IC). In brief, subjects rested for at least 15 min at room temperature. The BP cuff was placed around the upper left arm which was supported at the level of the heart while the subject sat in a chair with the feet positioned flat on the floor and with a straight back rested against the chair. The first measurement was taken after three minutes rest. The subject was told not to move or speak. In total four measurements were taken with one minute rest in between. The average of the last three measurements was calculated.

Clinical laboratory tests were performed at screening and at the end of treatment and included hematology, serum chemistry (liver enzymes, albumin, bilirubin, urea, creatinin, cholesterol parameters, triacylglycerols, glucose, minerals, blood sedimentation rate, and C-reactive protein). Blood samples were obtained from the antecubital vein of the forearm and collected in tubes containing clot activator for serum and in tubes containing potassium ethylene diamine tetra acid (K₃EDTA) for plasma (Vacutainer Systems, Becton Dickinson, Plymouth, UK). Blood was centrifuged for 15 min at ca. 2,000 × g at ca. 4°C within 15-30 min after collection, and stored at -20°C until analysis. All biochemical determinations in blood were performed at TNO Quality of Life using Olympus AU400 analytical equipment and reagents. Dipstick urinalysis was performed on the day of collection to assess the presence of protein, glucose, leucocytes, erythrocytes, nitrite, pH, ketones, bilirubin, and urobilinogen. A microscopic inspection of sediment was done if the dipstick test gave values above the normal range for leucocytes, blood or protein.

At the end of treatment, subjects indicated the presence or absence of a number of possible adverse effects in a questionnaire using a 4-point scale from 'not at all' to 'very often'. Symptoms included not feeling well, headache, weakness, fatigue, nausea, vomiting, burping, stomach-ache, bloating, flatulence, constipation, diarrhea, dry mouth, change of taste, cough, and skin complaints.

For renin, blood was collected in Vacutainer^® tubes containing K3EDTA as anticoagulant and centrifuged immediately (15 minutes at 2,000 × g and ca. 20°C). Plasma was snap frozen in liquid nitrogen and stored at -80°C. For angiotensin I and II, blood was collected in Vacutainer^® tubes containing K3EDTA as anticoagulant and protease inhibitor buffer was added at an amount of 50 μL buffer/ml blood. The buffer consisted of (stock 100 mL): 4250 mg EDTA, 8.13 mg enalaprilate, 200 mg neomycin, 0.5 mg o-phenantroline, 2% ethanol, 6 mg renin inhibitor (Sigma, cat.nr. C9415). Samples were centrifuged within 10 minutes after collection (15 minutes at 2,000 × g at 4°C) and plasma was snap frozen in liquid nitrogen and stored at -80°C.

Renin was measured by a standard immunoradiometric assay (Schering SA, CIS bio international, France). Intra- and inter-assay coefficients of variation were 4.1% and 7.6%, respectively. Angiotensin I and II were measured by a standard radioimmunoassay (Peninsula Laboratories, St. Helens, UK) following C-18 Sep-Pak (Waters-Millipore) extraction of the peptide. Intra- and inter-assay coefficients of variation were 4.6% and 7.7%, respectively.

For blood pressure, for every subject and treatment, measurements were first averaged per test day, and subsequently averaged over two successive test days. Statistical analysis of treatment effects on mean DBP and SBP and on mechanistic parameters was performed taking into account the study population, BP class (i.e. prehypertension or stage 1 hypertension), treatment, and interaction between treatment and BP class. Treatment effects were investigated using 2-way ANOVA. Carry over analysis was performed to determine whether a carry over correction was necessary. In case no significant carry over effect was found, the model was simplified by removing this effect. If the analysis of variance indicated an overall treatment effect (P < 0.05), comparisons between treatment means were performed using a 2-sided (paired) Student's t-test. Dichotomized scores in the questionnaire on possible adverse-effects were analyzed using Cochran-Mantel-Haenszel statistics taking into account multiple measurements per subject. In all statistical tests, the null hypothesis was rejected at the 0.05 level of probability (α = 5%). All data are presented as mean ± standard deviation (SD). Statistical analysis of the data was carried out using the SAS statistical software package (SAS/STAT Version 8.2, SAS Institute, Cary, NC).

Eighty healthy subjects (48 men, 32 women) were included with a mean age of 58 ± 8 y, body mass index (BMI) of 26.2 ± 3.1 kg/m2, systolic/diastolic BP of 136.2 (±12.7)/84.0 (±8.8) mm Hg. Thirty-four subjects had stage 1 hypertension with a mean systolic/diastolic BP of 147.0 (±11.7)/91.5 (±6.1) mm Hg, and 46 subjects had prehypertension with a mean systolic/diastolic BP of 128.1 (±6.1)/78.5 (±6.1) mm Hg. Upon data analysis, placebo BP values were found to be lower than baseline values at the time of screening. Because of this, a number of subjects fell into a lower BP class compared to the onset of the study. As there was no effect of treatment order on BP decrease of either intervention, subjects were classified as having prehypertension or stage 1 hypertension based on placebo values for further analysis of the treatment effects. There was no significant difference between treatments concerning the number of reclassified subjects. This procedure resulted in 26 subjects having stage 1 hypertension, 44 subjects having prehypertension, and 10 subjects having a normal BP (systolic BP < 120 mm Hg and diastolic BP < 80 mm Hg). These normotensives were excluded from further analyses, because the number of subjects was too small for a sound evaluation. Baseline characteristics of the study population (n = 70) are shown in Table 2. Compliance with intake of the study substances, assessed by counting returned capsules, was very good. Also compliance with study procedures with respect to maintenance of habitual diet and physical activity pattern was very good.

The BP values after 4 weeks treatment are presented in Table 3. The data indicate that in stage 1 hypertensive subjects, MPH1 induced a significant lowering of systolic BP by 3.8 mm Hg (P = 0.0080) and of diastolic BP by 2.3 mm Hg (P = 0.0065) compared with placebo. In subjects with prehypertension, the differences in systolic BP and diastolic BP between MPH1 and placebo were not significant. MPH2 did not change BP significantly as compared with placebo in either stage I hypertensives or prehypertensives.

Intake of both MPHs was well tolerated. No significant differences in clinical laboratory parameters and adverse events were observed between placebo and MPHs.

Thirty seven subjects reported a adverse effect after intake of the placebo (in total 118 occurrences of adverse effects), 43 subjects reported a adverse effect after intake of MPH1 (in total 126 occurrences of adverse effects), and 37 subjects reported a adverse effect after consumption of MPH2 (in total 114 occurrences of adverse effects). Most reported symptoms occurred 'hardly ever' or 'sometimes'. In single occasions, a symptom occurred 'often' or 'very often', but no clear differences were observed between treatments. Adverse effects most often reported were flatulence, headache, dry mouth, and fatigue. Only flatulence was significantly more often reported after placebo treatment (23×) compared with MPH1 (16×; P = 0.0196) and MPH2 (14×; P = 0.0339) (Figure 1). Since the frequency and seriousness of the reported adverse effects was low, the above mentioned differences between MPHs and placebo were not considered clinically relevant.

No significant differences between MPHs and placebo treatment were found in plasma renin activity or concentrations of angiotensin I and II (Figure 2). In all subjects, intake of the placebo, MPH1, and MPH2 for 4 weeks resulted in similar mean renin activities. Mean angiotensin I and II concentrations after intake of placebo were comparable with those after intake of MPH1 and MPH2.