Dose‐dependent lowering of blood pressure by dairy peptides in mildly hypertensive subjects

Abstract

Clinical studies have demonstrated a beneficial effect on blood pressure for milk derived material containing isoleucyl‐prolyl‐proline (IPP) and valine‐prolyl‐proline (VPP) peptides. The aim of the present study was to evaluate the blood pressure lowering effect of three different IPP and VPP doses in products with a comparable electrolyte and protein composition. The present study was designed as a randomized, double‐blind, parallel‐group, dose–response trial: 166 subjects (> 140/90 mmHg) received placebo during a 2‐week run‐in, 8‐weeks intervention followed by a 2‐week washout. Results indicate that materials containing IPP and VPP do lower blood pressure dose‐dependently (p < 0.05 for diastolic blood pressure, DBP). The effect on systolic blood pressure (SBP)/DBP over 8 weeks compared with placebo was + 0.1/− 1.3, − 1.5/− 1.4 and − 2.5/− 1.9 mmHg for the low, medium and high dose of peptides, respectively. The percentages of subjects who showed a fall in SBP > 3 mmHg or who attained an SBP below 140 mmHg, were 54% (placebo), 64% (low), 76% (medium) and 71% (high dose) respectively. This effect can only be demonstrated for office pressure and not for home or ambulatory pressure. Furthermore, the results suggest that the magnitude of the fall in blood pressure is a function of baseline blood pressure. We conclude that IPP and VPP may have a modest dose‐dependent effect on office blood pressure in mildly hypertensive subjects although this could not be confirmed with ambulatory or home blood pressure measurements.

• Blood pressure
• dairy peptides
• IPP
• VPP

Introduction

Recommendations about lifestyle form an integral part of the (initial) management of hypertension [1]. Except for moderate exercise, quitting smoking and reducing alcohol intake, these recommendations include dietary advice. Based on the results of the DASH‐trial, one may consider prescribing a diet rich in fruits and vegetables, and low in saturated fats and cholesterol [2]. In addition, sour milk and other dairy products may lower the pressure. For instance, a number of studies indicate that the consumption of bioactive peptides is associated with a fall in blood pressure of approximately 6–9 mmHg systolic (SBP) and 3–7 mmHg diastolic (DBP) [3–8]. Although the products that have been tested contain several peptides which could be responsible for this effect, the lactotripeptides (LTPs) valine‐prolyl‐proline (VPP) and isoleucyl‐prolyl‐proline (IPP) are of particular interest because these two peptides carry a high angiotensin‐converting enzyme (ACE) inhibitory effect in vitro [9].

So far, most of the placebo‐controlled human studies with dairy products containing VPP and IPP have been performed in Japanese and Finnish hypertensive patients only [3–8], [10]. In all but one of these, a parallel group design was used and in some of these studies, patients who participated were taking antihypertensive medication during the trial [5], [6]. One study from Finland evaluated untreated patients according to a crossover design [10]. Blood pressure responses varied from rather modest to impressive. Up until now, only one study has addressed the dose–response relationship at relatively low levels of IPP and VPP in Japanese subjects [7]. In all other studies, fixed doses of the peptides were used. Moreover, the product that was used in the Finnish trials contained more electrolytes than that in the previous studies, which could have contributed to the blood pressure changes. Since in these earlier studies differences in the magnitude of the effect between the Japanese and the Finnish subjects were observed, we set up the present trial to evaluate whether the LTPs VPP and IPP, when given in different dosages but with a comparable electrolyte composition, would be able to reduce blood pressure and ACE activity in a dose‐dependent manner in other European subjects. The effect on office, home and ambulatory blood pressure was evaluated over an 8‐week intervention period.

Patients and methods

Patients

Participants for this study were recruited by advertisements in local newspapers. The advertisement only mentioned the purpose of the trial and did not mention any inclusion qualifications other than a mildly elevated blood pressure. A total of 579 subjects responded and were screened for eligibility at two visits, at least one day apart. Respondents who used antihypertensive drugs and in whom treatment could be safely withheld were asked to first stop their medication for a period of 3 weeks before the qualifying visits. At the first screening visit, we took three measurements of blood pressure using a validated automatic oscillometric device (Omron HEM 907) after 15 min of rest. Only non‐smoking subjects with an average SBP between 140 and 190 mmHg and an average DBP equal to or below 110 mmHg (without treatment) were invited to the second visit. When they had a SBP between 140 and 180 mmHg and a DBP equal to or below 100 mmHg at the second visit, they qualified for the study proper. Further requirements for inclusion were age between 35 and 70 years and a body mass index (BMI) of at least 18 kg/m² but not more than 32 kg/m². In addition, biochemical analysis of blood and urine had to be normal. Exclusion criteria were pregnancy, alleged intolerance or allergy to dairy products, and the use of medications or substances that could interfere with the bioavailability of the test product. The study was approved by the local Medical Ethics Committee and all participants gave their informed consent in writing.

lStudy design

The study was designed as a randomized, double‐blind, placebo‐controlled, parallel‐group trial, in which patients received either placebo or one of three doses of the test product (see below). Subjects were randomized over the various treatment groups in blocks according to their baseline office SBP (week 0) and stratified for age, weight and sex. Following randomization, eligible patients first entered a run‐in period of 2 weeks, during which they received placebo drink (200 ml daily). Thereafter, they consumed either placebo or the test product for 8 weeks. This was followed by a 2‐week washout period without any drink. Subjects were instructed to consume the product daily in the morning with breakfast. Every 2 weeks, subjects visited the clinic where body weight and blood pressure were measured. To assess compliance, participants were asked to return used and unused cups on each visit. Subjects were advised not to alter their usual diet and lifestyle.

Before and at the end of the intervention, participants collected 24‐h urine for determination of sodium, potassium, creatinine and micro‐albumin. Blood was drawn at baseline and again at week 4, week 8 and week 10 for determination of ACE activity as well as levels of active renin concentration (APRC). ACE and APRC were measured according to standard methods [11], [12].

Composition of test and control products

All test products were pasteurized yoghurt drinks manufactured by Unilever. The test article is LTP powder, which is prepared through enzymatic hydrolysis of the milk protein, casein. A placebo and three test products with increasing amounts of LTP (2.3, 4.6 and 9 mg/200 g) were used. This resulted in consumed IPP and VPP levels of 1.13 mg/200 g and 1.17 mg/200 g for the low‐dose LTP product, 2.30 mg/200 g and 2.26 mg/200 g for the medium dose and 4.56 mg/200 g and 4.47 mg/200 g for the high dose respectively. The addition of different dosages of casein hydrolysate in the test products led to an extra protein content, which was corrected for with skimmed milk powder. Consequently, the nutritional composition of all products was 13% carbohydrate, 0.25% fat, 3.4% protein and 130 mg/100 g calcium. Pectin was added for stability and sugar, and flavour for taste.

Test products were analysed for full composition at the start and the end of the intervention periods. During the trial, products were stored at −20°C at the research site and between +4°C and −4°C in the refrigerator at the subjects’ home.

Blood pressure measurements

At each visit, office blood pressure measurements were collected. Office blood pressure (OBP) was taken after 15 min of rest with an oscillometric automatic device (Omron HEM 907) approximately 2.5–3 h after intake of the test product. Each measurement comprised three successive readings obtained within 10 min; the mean of the last two was used for analysis. In the same weeks that patients came in for their office blood pressure measurements as well as on the first day of the intervention, they also took their pressures at home. With respect to these home blood pressure measurements (HBP), patients were instructed to measure their blood pressure after 15 min of rest before breakfast as well as 3 and 6 h after intake of the test product. At each occasion, patients collected three measurements within 10 min. Again, only the last two were averaged and used for analysis. Patients used an automatic device that saved the data in memory but left the observer blinded.

Except for OBP and HBP, we also measured 24‐h ambulatory blood pressure (ABP) at baseline (week 0) and after 8 weeks of treatment. ABP was measured using the Spacelabs monitor type 90217 at the non‐dominant arm. Readings were taken every 15 min during the day and every 30 min during the night.

Statistical considerations

The primary outcome of this study was the difference between the average SBP at week 8 and that at baseline for the various treatments. Secondary outcomes included DBP and heart rate at each time point. Based on an earlier work we estimated the long‐term variance (i.e. over the 8‐week period) of SBP to be 6 mmHg. We calculated that 40 subjects per treatment group would be sufficient to detect a change in SBP of 5 mmHg with a power of 80% and alpha;=0.05 (one sided). To allow for dropouts during the study, we recruited 42 subjects per treatment group, yielding a total of 168 subjects to be included.

Data were analysed using ANOVA for repeated measurements and SPSS version 12 as the statistical package. Data are expressed as means with standard deviations (SD).

Results

Of the 579 subjects who responded to our advertisement, 394 did not pass the screening phase either because their blood pressure remained outside the pre‐specified limits (n=281) or because there were other medical reasons that could interfere with the study (n=61). Another 52 subjects did not want to enter the study after having been informed about the nature of the investigations. The remaining 185 participants were randomized and entered the intervention phase of the trial. Clinical characteristics of the subjects who were randomized did not differ from those of the initial population. Subjects entered the study in two cohorts. The first cohort was followed from April until August and the second one from September until January. During the study, another 19 patients dropped out. Reasons for dropping out were non‐specific adverse events (n=14) or personal problems (n=4). One subject continued to take antihypertensive medication and was taken out of the trial. Thus, a total of 166 persons completed the trial. There were no differences in clinical characteristics between those who completed the trial and those who dropped out.

Office blood pressure measurements

Baseline characteristics for each group are given in Table I; the four groups were well matched both with regard to baseline office blood pressure and other variables. Baseline blood pressure did not differ between the two cohorts. Although SBP fell in all four groups during the first half of the intervention part of the trial, the fall in pressure was greater in those with the medium and the high dose of the dairy drink than in the low‐dose group (Figure 1). A significant fall in DBP was observed at weeks 2 and 4 for those who consumed the high‐dose LTP. The medium dose of LTP decreased DBP only after 2 weeks of intervention. When the results over 8 weeks were corrected for the placebo response, only the high‐dose group displayed an almost significant fall in DBP (p=0.078; Figure 2). However, a clear dose‐dependent blood pressure lowering effect was observed, which was significant for DBP (p<0.05). Although there was no significant interaction between dose and cohort, differences in blood pressure between placebo and treatment tended to be somewhat greater in the second cohort (i.e. during the autumn and winter period).

Table I. Clinical characteristics of the patients at the time of randomization (mean ± SD).

	Placebo	Low dose	Medium dose	High dose
Age (years)	59±7	58±8	58±7	60±7
Gender (M/F)	22/19	22/20	26/16	21/20
Weight (kg)	79±13	77±12	81±13	79±12
SBP (mmHg)	148±13	147±12	146±14	149±15
DBP (mmHg)	85±9	85±7	85±10	85±10
HR (beats/min)	68±9	69±9	70±10	67±7
Serum creatinine (μmol/l)	79±13	79±13	80±13	79±12
Plasma ACE (U/l)	17.0±5.2	16.2±4.6	16.9±4.8	17.2±4.6
UnaV (mmol/24 h)	175±65	154±48	164±60	180±92

M, male; F, female; SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate; ACE, angiotensin-converting enzyme; UnaV, urinary sodium excretion.

Figure 1. Time course of blood pressure during the various phases of the trial. SBP, Systolic blood pressure; DBP, Diastolic blood pressure; LTP, Lactotripeptides.

Figure 2. Time course of blood pressure during the various phases of the trial. SBP, Systolic blood pressure; DBP, Diastolic blood pressure; LTP, Lactotripeptides.

When we calculated the proportion of patients in whom SBP fell by at least 3 mmHg or who attained a SBP below 140 mmHg, response rates were 54%, 64%, 76% and 71% for the placebo, low‐, medium‐ and high‐dose groups respectively. Again, more of those ‘responders’ were observed in the medium‐ and high‐dose LTP group compared with the low‐dose LTP and placebo group (74% versus 59%, p<0.05 for trend). During the washout period (from week 8 to week 10), both SBP and DBP returned to their respective pre‐treatment levels (Figures 1 and 2). Heart rate was not affected by any of the treatments or at any time period. Not unexpectedly, patients with the highest pressures at baseline showed the greatest response, regardless of dose. In multivariate analysis, baseline SBP and dose of the test product appeared to be the only predictors of the fall in SBP during treatment.

Home blood pressure and 24‐h ambulatory blood pressure

HBP on the first day of the intervention did not reveal any differences in blood pressure changes between groups. The same was true when blood pressure changes during and after the intervention were considered. ABPM data at baseline an after treatment also failed to disclose any differences between the groups, whether analysed for the entire 24 h or for the daytime and night‐time periods separately.

Laboratory assessments

Body weight did not change in any of the four groups. Urinary sodium, potassium and creatinine excretion remained constant during the trial and did not differ between groups at any time point. None of the treatments had any influence on micro‐albumin excretion, which was variable but not different between groups. ACE activity as well as APRC concentrations did not differ between groups and remained unaltered during the treatments.

Discussion

The present study shows that a dairy drink containing the lactotripeptdes VPP and IPP is able to modestly lower office blood pressure in a dose‐dependent way. Previously, two other products containing these peptides have been evaluated for their antihypertensive potential. One of these was manufactured in Japan (Calpis®, Calpis Food Industry Co., Ltd., Tokyo, Japan). This is a sour milk, which has been fermented with Lactobacillus helveticus and Saccharomyces cerevisiae [9]. The other product is Evolus® (Valio Ltd., Helsinki, Finland), which is produced by fermentation with Lactobacillus helveticus LBK‐16H [10]. In spontaneously hypertensive rats (SHR), single oral administration of either Calpis® sour milk or the peptides VPP and IPP in corresponding units significantly reduces SBP for several hours [9]. With long‐term treatment, the development of high blood pressure is even significantly attenuated [13], [14]. Remarkably, though, no effect of the tripeptides is seen in control normotensive Wistar–Kyoto rats [9] suggesting that the products only work when blood pressure is elevated. Additional experiments using infusions of angiotensin I and II provided evidence for an ACE‐inhibiting action of the sour milk product [13], [14].

In humans, VPP and IPP also lower blood pressure. For instance, Hata and coworkers [5] conducted a placebo‐controlled trial in 36 elderly hypertensive patients who were given either placebo (artificially acidified milk) or a fixed amount of Calpis® sour milk (95 ml, containing 1.5 mg of VPP and 1.1 mg of IPP) for a period of 8 weeks. In the sour‐milk group, SBP fell significantly at 4 and 8 weeks of treatment, by 9.4 ±3.6 mmHg (mean±SEM, p<0.05) and 14.1±3.1 mmHg (p<0.01), respectively. DBP was also significantly reduced. No changes in blood pressure were observed in the placebo group. It should be noted, however, that most patients were taking antihypertensive drugs and that blood pressure was measured only once at each visit. Another problem with this study is that six patients did not complete the trial but no intention‐to‐treat analysis was performed. All these factors make it a bit difficult to appreciate fully the effects of the milk preparation. However, other studies have confirmed that VPP and IPP also lower blood pressure in untreated hypertensives [3], [4], [7], [8], [10], [15]. In a recent randomized, placebo‐controlled, double‐blind study in 40 subjects with high‐normal blood pressure (HN group) and 40 subjects with mild hypertension (MH group), each subject ingested six test tablets daily (12 g) containing either powdered milk that had been fermented with L. helveticus CM4 or the same amount of placebo [15]. Compared with placebo, 4 weeks of treatment with the test product reduced SBP by an average of 11.2 mmHg (p=0.003) in the MH group but by only 3.2 mmHg (p=0.27) in the HN group. For DBP, these figures were 6.5 mmHg (p=0.055) and 5 mmHg (p=0.04) in MH and HN, respectively). Likewise, Mizuno and associates [7] found that a VPP/IPP product lowered blood pressure in patients with mild hypertension but not in those with high‐normal blood pressure [7]. These data suggest that the dairy peptides have a greater effect in patients with higher blood pressure levels.

In Europe, Finnish investigators have evaluated the effects of Evolus® on blood pressure. Using a fixed dose of this product, Seppo and co‐workers [6] demonstrated in 39 hypertensive patients, 16 of whom used antihypertensive medication, that L. helveticus LBK‐16H fermented milk significantly lowered blood pressure over a 21‐week period. Interestingly, self‐measured rather than office blood pressures were used for analysis. The same group later showed that the milk product that contained 2.4–2.7 mg of VPP and 2.4–2.7 mg of IPP also lowered office pressure in a group of 60 untreated hypertensives [10]. However, in that particular study, the fall in SBP was rather small (2 mmHg more with the active product than with placebo) while changes in DBP with Evolus® did not differ from those on placebo. This is remarkable because Evolus® contains a higher amount of the tripeptides than the Japanese product. Overall, the European studies suggest a smaller blood pressure reduction than the Japanese studies. This is confirmed by our study. One can only speculate about possible explanations such as race and background diets, but it would be of interest to evaluate this in greater detail.

Mizuno and coworkers [7] also examined the effect of different dose levels of the tripeptide containing casein hydrolysate. A total of 131 volunteers with high‐normal blood pressure and mild‐ hypertension were randomly divided into four groups. Each volunteer was given two tablets, containing four different dosages of VPP and IPP (VPP+IPP: 0, 1.8, 2.5 and 3.6 mg), daily for 6 weeks. A significant decrease in SBP was observed after 6 weeks in all the active groups compared with SBP measured before treatment. Changes in the SBP after 6 weeks of treatment in the four groups were −1.7, −6.3, −6.7 and −10.1 mmHg, and these effects were dose‐dependent. In addition, a significant difference in SBP between the placebo group and the VPP and IPP group receiving 3.6 mg was observed (p<0.001).

Our study differs from the previous ones in several aspects. We not only measured office pressure but also home and 24‐h ambulatory blood pressure. Furthermore, in view of the alleged ACE‐inhibitory action of the tripeptides, we measured ACE activity in plasma as well. The present data indicate that home and ambulatory pressures do not show a significant hypotensive response to the LTPs. This does not necessarily mean that there is no effect at all, since a growing body of evidence suggests that office, home and ambulatory pressures carry different aspects of information regarding the cardiovascular system (17,18). Certainly, our data with respect to office pressures seem to suggest that the product lowers blood pressure dose‐dependently although the absolute fall in pressure is still rather modest. When corrected for the placebo response, the decrement in blood pressure was even only of borderline statistical significance. Although ambulatory and home blood pressure measurements may be superior to office blood pressure, we have to realize that office pressure and not ambulatory or home pressure was the primary outcome variable in this study. Because office pressures tend to be higher than either home or ambulatory pressures, our results may still be in line with previous studies, which showed that the effect of the tripeptide products is easier to demonstrate when blood pressure is higher. Indeed, the data from the present study suggest that the magnitude of the fall in SBP is a function of baseline SBP. It is also possible that the effect of the tripeptides is only short‐term so that it may be detected with office measurements (a few hours after ingestion) but not with measurements over a longer period. If such would be the case, then perhaps a single dose is not sufficient to produce significant changes in 24‐h blood pressure. Finally, we cannot entirely exclude the possibility that subjects from the placebo group ingested some bioactive peptides from other, natural sources. If this were the case, this would dilute the difference between the placebo and the other groups.

Although the LTPs have been shown to possess ACE‐inhibitory activity in vitro, we did not find any decrease in plasmatic ACE, not even with the highest dose, which lowered blood pressure. Admittedly, this does not entirely exclude an ACE‐inhibiting mechanism of action since inhibition may have been achieved only at the tissue level. Nevertheless, renin levels did not change either, something that one would expect to occur when adequate ACE‐inhibition had been achieved. Thus, more evidence is needed before we can conclude that the compound lowers the pressure in man by interfering with the rennin–angiotensin system. Alternatively, LTPs may affect blood pressure through a variety of other mechanisms, including inhibition of sympathetic activity or the liberation of vasoactive substances from the gut. More mechanistic studies will be necessary to assess these possibilities.

We conclude that a dairy drink containing the peptides VPP and IPP causes only a modest but presumably dose‐dependent reduction in office blood pressure and more so when baseline pressure is higher. However, because this finding is not corroborated by our data on ambulatory and home pressure measurements, more and larger studies or perhaps a meta‐analysis of existing data are necessary to establish whether VPP and IPP truly lower blood pressure.

Acknowledgements

This study was supported by a grant from Unilever Food & Health Research Institute, Vlaardingen, The Netherlands.

Conflict of interest: Kim van der Zander and Mettina M.G. Koning are employees of Unilever Food & Health Research Institute, Vlaardingen, The Netherlands.