Effects of Val-Pro-Pro and Ile-Pro-Pro on Nondipper Patients: A Preliminary Study

Abstract

Much clinical evidence on the antihypertensive effects of the milk-derived antihypertensive peptides Val-Pro-Pro and Ile-Pro-Pro (lactotripeptides) has been reported. However, circadian rhythm effects determined by ambulatory blood pressure monitoring (ABPM) to eliminate the confounding influence of the white-coat effect have not been fully studied. Twelve hypertensive patients not receiving antihypertensive medication (2 men, 10 women; mean age±standard deviation, 63.5±8.3 years) who had been visiting our clinic for more than 1 year participated in this study. Mean (±standard deviation) systolic blood pressure (SBP) and diastolic blood pressure (DBP) were 142.4±2.6 and 83.5±6.4 mm Hg, respectively, at the first office visit. After patients ingested a fermented milk product containing antihypertensive peptides (2.53 mg Val-Pro-Pro; 1.52 mg Ile-Pro-Pro) for more than 4 weeks, both office SBP and DBP were significantly reduced to a mean (±standard deviation) of 133.3±7.0 mm Hg and 76.5±8.4 mm Hg (P<.001 and P<.005 by paired t-test), respectively. The 24-hour SBP and DBP determined by ABPM were reduced from 127.3±2.4 and 78.7±2.3 mm Hg to 120.2±2.4 and 75.0±2.2 mm Hg (P<.001 and P<.05), respectively. Awake-time SBP (08:00–21:00), night-time SBP (0:00–05:00), and early-morning SBP (06:00–07:00) were reduced from 130.9±2.4 to 123.3±2.3 mm Hg, 118.7±2.9 to 113.2±3.4 mm Hg, and 132.8±4.3 to 122.4±3.9 mm Hg (by paired t-test: P<.001, P<.05, and P<.05), respectively. As seen with DBP measured by ABPM, 24-hour DBP and awake-time DBP were significantly reduced from 78.7±2.3 to 75.0±2.2 mm Hg and 82.1±2.5 to 77.3±2.2 mm Hg (P<.05 and P<.01), respectively. Office BP and 24-hour blood pressure did not significantly differ between the dipper and nondipper groups at baseline. However, after treatment, night-time and early-morning blood pressure were significantly reduced from baseline in the nondipper group (−8.5±2.5 and −15.6±3.7 mm Hg; P<.05 and P<.01, respectively) but not in the dipper group (−2.5±3.6 and −1.2±4.7 mm Hg; P not significant), and the reduction in early-morning blood pressure significantly differed between the groups (P<.05). These results suggest that Val-Pro-Pro and Ile-Pro-Pro decrease blood pressure in patients with stage I hypertension and result not only in lower blood pressure at night-time but also in lower early-morning SBP in nondipper patients.

Introduction

H ypertension is an important risk factor for all major atherosclerotic cardiovascular disease outcomes, including cardiac failure, stroke, coronary artery disease, and peripheral vascular disease.¹ Use of antihypertensive medications to achieve a normal blood pressure range in hypertensive patients and recommendations for lifestyle changes in patients with prehypertension are very important for reducing the incidence of cardiovascular events.² Lifestyle modification by means of weight loss, regular exercise, and low alcohol consumption are likely to reduce blood pressure in prehypertensive patients, allowing avoidance of the use of antihypertensive drugs. Sodium restriction and stress management are also important in blood pressure management. However, continuously controlling diet, regularly exercising, and avoiding or successfully managing stress are typically not easy to accomplish.

Certain milk peptides with antihypertensive effects putatively linked to their angiotensin-converting enzyme (ACE) inhibitory activities have been reported^3,4 and reviewed.^5,6 Two kinds of milk-derived peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), first isolated from milk fermented with Lactobacillus helveticus,^4,7 have been extensively studied in many clinical trials in Japanese^8
–10 and Finnish^11,12 patients and have been reviewed.^8

–14 However, placebo effects during the pretreatment period were observed in some studies, resulting in no detection of significant effects during treatment with VPP and IPP.^15,16 Given these findings, the importance of precise monitoring of blood pressure and careful selection of volunteers for the detection of lactotripeptide effects may reduce placebo effects.

In addition, ambulatory blood pressure monitoring (ABPM) has many advantages over clinical measurements to avoid the confounding influence of the white-coat effect.¹⁷ Variations during ABPM appeared to be more closely associated with cardiovascular events than casual blood pressure measurements in patients with essential hypertension.¹⁸ The circadian pattern of blood pressure determined by ABPM suggests a significantly higher cardiac vascular risk in nondippers (people whose blood pressure does not decrease at night) than in dippers (people whose blood pressure decreases at night).¹⁹ Information on blood pressure in the morning and at night is especially important in predicting cardiac events, such as stroke and myocardial infarction.

Clinical trials using ABPM to detect whether the food-derived peptides VPP and IPP affect blood pressure have been performed.^11,12,20,21 However, evidence of circadian rhythm effects at night and early morning, and dipper and nondipper effects are not well understood. In the present study, patients who had consulted with a family physician in our clinic for more than 1 year were recruited. The rationale was that selection of patients who were familiar with both the physician and the clinic would help to avoid the white-coat effect and achieve good adherence. In this small, preliminary study, ABPM was used to analyze changes in circadian patterns by intake of VPP and IPP.

Materials And Methods

Participants

Twelve patients who had previously visited the Iketani Clinic in Tokyo, Japan, to consult with their family physician, were diagnosed as having stage I hypertension (systolic blood pressure [SBP] of 140–159 mm Hg or diastolic [DBP] of 90–99 mm Hg), but were not taking antihypertensive medications were enrolled in the study. Patients with severe hypertension, allergic asthma, severe liver or renal diseases, stroke, and myocardial infarction were excluded. This trial was done in accordance with the Declaration of Helsinki after informed consent from all patients had been obtained during the initial or follow-up visit.

Study protocol and office blood pressure measurement

The study was a small, nonparallel trial performed in a single clinical center. Patients were instructed to visit the Iketani Clinic in the morning to have a diagnostic interview, and brachial blood pressure was measured in the clinic by a family physician with an automated sphygmomanometer (HEM-907; Omron, Kyoto, Japan) after the patients had rested for 5–10 minutes in a sitting position. At the first clinic visit, the patients were informed about the study. Brachial SBP and DBP were measured in all study participants by an automated sphygmomanometer. Three measurements were taken at 1-minute intervals with patients in the sitting position, and the lowest value was used to define clinic SBP and DBP. Table 1 shows the clinical characteristics of these patients at the time of this first examination. These patients were instructed not to make any lifestyle changes and not to take antihypertensive agents throughout the test period (about 4 weeks). All patients were instructed to take a commercially available fermented milk product (160 g) containing antihypertensive peptides—2.35 mg of VPP and 1.52 mg of IPP—every morning for about 1 month. The amounts of VPP and IPP in the test sample were measured by the liquid chromatography-mass spectrometry method as described elsewhere.²² The product was processed from fermented milk with L. helveticus, and its composition is listed in Table 2.

Table 1.

Patient Characteristics

Characteristic	Value
Men (n)	2
Women (n)	10 (3 with hyperlipidemia)
Age (y)	63.5±8.3
Office systolic blood pressure (mm Hg)	142.4±2.6
Office diastolic blood pressure (mm Hg)	83.5±6.4
Office pulse rate (beats/min)	69.0±12.5

Values expressed with a plus/minus sign are the mean±standard deviation.

Table 2.

Composition of Test Sample (per 100 g)

Substance	Amount
Protein (g)	1.3
Carbohydrate (g)	3.4
Ash (g)	0.4
Fat (g)	0
Fiber (g)	0.7
Moisture (g)	94.2
Maltitol (g)	1.1
Energy (kJ)	71
Potassium (mg)	68
Calcium (mg)	49
Phosphorus (mg)	39
Sodium (mg)	70
Val-Pro-Pro (mg)	1.58
Ile-Pro-Pro (mg)	0.95

ABPM

One day before the start of the treatment and 1 day after completion of the approximately 1-month treatment period, ABPM was conducted for 24 hours. All data obtained at the end of the treatment were mixed and calculated as the post-treatment results. ABPM was performed by using an ambulatory blood pressure measurement device (ES-H531; Terumo, Tokyo, Japan) on the nondominant arm. The device was set to obtain blood pressure and heart rate readings at 30-minute intervals during the day (08:00–24:00) and night (24:00–08:00). All patients were asked to go to bed no later than 23:00 and not to exercise or expend more than the usual amount of activity. The recording was then analyzed to obtain 24-hour, awake-time (08:00–21:00), night-time (0:00–05:00), and early-morning (06:00–07:00) information on SBP, DBP, and heart rates. Nocturnal dipping was defined as a reduction in the average SBP at night greater than 10% compared with average daytime values.

Statistical analysis

All values are expressed as means±standard errors. Changes in ABPM values before and after treatment were analyzed by use of JLM software (SAS Institute, Cary, NC, USA). Changes in SBP, DBP, and heart rate for 24 hours and during awake time, night-time, and early morning monitored every 30 minutes were statistically analyzed by using the paired t-test. Changes between the dipper group and the nondipper group were statistically analyzed by using the Student t-test. Values with a P value less than .05 according to a 2-tailed test were considered to be statistically significant.

Results

The 12 participants (2 men, 10 women) had a mean age of 63.5±8.3 years, a mean pretreatment SBP of 142.4±2.6 mm Hg, and a mean pretreatment DBP of 83.5±6.4 mm Hg (±standard deviations) (Table 1). All patients took the fermented milk product every day for about 1 month (average period, 33.0±12.7 days). The averages of the office SBP and office DBP at the last visit after the treatment were 133.3±7.0 mm Hg and 76.5±8.4 mm Hg (P<.001 and P<.005, respectively, compared with baseline). SBP and DBP obtained by ABPM (24-hour blood pressure) had changed significantly from 127.3±2.4 to 120.2±2.4 mm Hg and from 78.7±2.3 to 75.0±2.2 mm Hg (P<.001 and P<.05), respectively. Awake-time SBP and DBP (08:00–21:00) had also changed significantly from 130.9±2.4 to 123.3±2.3 mm Hg and from 82.1±2.5 to 77.3±2.2 mm Hg (P<.001 and P<.01), respectively. Night-time SBP had significantly changed from 118.7±2.9 to 113.2±3.4 mm Hg after treatment with VPP and IPP (P<.05). Early-morning SBP had changed from 132.8±4.3 to 124.4±3.9 mm Hg (P<.05). Table 3 summarizes these results.

Table 3.

Changes in Blood Pressure and Pulse Rate Before and After Intake of Fermented Milk Containing Val-Pro-Pro and Ile-Pro-Pro

Variable	Before Intake	After Intake	P Value ^a
Office BP (mm Hg)
SBP	142.4±2.6	133.3±7.0	<.001
DBP	83.5±6.4	76.5±8.4	<.005
24-hour BP (mm Hg)
SBP	127.3±2.4	120.2±2.4	<.001
DBP	78.7±2.3	75.0±2.2	<.05
Awake time (08:00–21:00) BP (mm Hg)
SBP	130.9±2.4	123.3±2.3	<.001
DBP	82.1±2.5	77.3±2.2	<.01
Night-time (0:00–05:00) BP (mm Hg)
SBP	118.7±2.9	113.2±3.4	<.05
DBP	72.2±2.1	70.3±2.7	NS
Early-morning (06:00–07:00) BP (mm Hg)
SBP	132.8±4.3	122.4±3.9	<.05
DBP	81.4±3.0	78.4±3.1	NS
Pulse rate (beats/min)
24-hour	66.7±3.0	65.8±2.6	NS
Awake time (08:00–21:00)	69.8±2.9	69.6±3.0	NS
Night-time (0:00–05:00)	62.6±3.2	62.0±2.9	NS

Unless otherwise noted, values are expressed as the mean±standard error.

Significant difference between before and after treatment.

BP, blood pressure; DBP, diastolic blood pressure; NS, not significant; SBP, systolic blood pressure.

For a more thorough understanding of the circadian pattern of blood pressure, SBP and DBP obtained by ABPM were analyzed after patients were divided into dipper and nondipper categories (Table 4). Office blood pressure and 24-hour blood pressure did not significantly differ between the dipper and the nondipper groups at baseline (data not shown). However, of interest, night-time SBP and early-morning SBP in the nondipper group were significantly reduced from the baseline, whereas those values in the dipper group were not. Post-treatment reduction in early-morning SBP was significantly different between the nondipper group (change, −15.6±3.7 mm Hg; P<.05) and the dipper group (change, −1.2±4.7 mm Hg; P not significant), as shown in Table 4. These results suggest that VPP and IPP can decrease blood pressure in patients with stage I hypertension and can also reduce not only night-time but also early-morning SBP in nondipper patients.

Table 4.

Changes in Blood Pressure and Pulse Rate Before and After Ingestion of Fermented Milk containing Val-Pro-Pro and Ile-Pro-Pro According to Dipper vs. Nondipper Status

	Dipper (n=6)				Nondipper (n=6)
Variable	Before Intake	After Intake	P Value ^a	Change	Before Intake	After Intake	P Value ^a	Change
Office BP (mm Hg)
SBP	143.7±2.3	132.3±8.3	<.05	−11.3±7.7	141.2±2.4	134.2±5.9	<.05	−7.0±6.2
DBP	81.7±7.7	75.5±7.1	<.01	−6.2±2.2	85.3±4.8	77.5±10.1	<.1	−7.8±9.5
24-hour BP (mm Hg)
SBP	127.9±3.1	121.1±3.8	<.05	−6.7±2.2	126.8±4.0	119.3±4.2	<.05	−7.5±2.1
DBP	80.0±3.4	75.8±3.6	<.05	−4.3±1.7	77.5±3.3	74.3±3.5	NS	−3.3±2.2
Awake-time (08:00–21:00) BP (mm Hg)
SBP	133.2±3.5	125±3.0	<.01	−8.4±1.7	128.7±3.5	121.7±3.7	<.1	−7.0±3.2
DBP	83.8±3.9	78.8±3.5	<.05	−5.5±1.7	80.4±3.3	75.8±3.2	<.1	−4.5±2.2
Night-time (0:00–05:00) BP (mm Hg)
SBP	115.6±2.4	113.2±5.0	NS	−2.5±3.6	121.8±5.3	113.3±6.2	<.05	−8.5±2.5
DBP	72.2±2.7	69.7±4.0	NS	−2.5±2.3	72.2±3.6	70.9±4.6	NS	−1.3±2.9
Early-morning (06:00–07:00) BP (mm Hg)
SBP	129.6±6.0	128.4±4.0	NS	−1.2±4.7	136.0±6.3	120.4±7.3	<.01	−15.6±3.7^b
DBP	80.7±4.8	79.6±4.5	NS	−1.1±5.1	82.2±4.1	77.1±5.1	NS	−5.1±2.8

Significant difference between before and after treatment.

Significant difference between dipper and nondipper group.

BP, blood pressure; DBP, diastolic blood pressure; NS, not significant; SBP, systolic blood pressure.

The 24-hour, awake-time, and night-time heart rates did not differ after treatment (Table 3). During the treatment period, no adverse events, such as the dry cough that has been reported with ACE inhibitors or gastrointestinal symptoms, were observed.

Discussion

Many reports have shown a significant lowering of blood pressure in hypertensive patients after repeated intake of the food (casein)-derived peptides VPP and IPP for a few weeks.^8

–14 Management of BP measurement is very important in detecting the effects of lactotripeptide intake, which might be very mild because of a placebo effect,¹⁵ and the confounding influence of the white-coat effect has sometimes concealed these effects in clinical studies. An ABPM device, which is often used to overcome the white-coat effect, was applied in clinical studies in Finnish,^11,12 U.S.,²⁰ and Japanese²¹ patients, and some reported significant effects of VPP and IPP.^12,20,21 In this study, we noted significant changes not only in the office blood pressure but also in some ABPM blood pressure values after intake of VPP and IPP for about 4 weeks; these findings were seen even though this study was small and lacked a placebo group. Patients who had a history of visiting our clinic and had a trusting relationship with our family physicians for more than 1 year seemed to be appropriate to select as volunteers. For a more detailed understanding of the circadian pattern of blood pressure, a large placebo-controlled parallel study conducted among a network of family physicians using their own patients as participants would be necessary in the future.

Use of ABPM in patients with a long history of medical care at our clinic revealed a significant effect of VPP and IPP on awake-time blood pressure and night-time blood pressure. Of interest, results of the comparison of blood pressure between the dipper and nondipper groups suggested greater effectiveness in reducing night-time (0:00–05:00) and early-morning (06:00–07:00) SBP in the nondipper group. Nondippers have a high risk for vascular complications, particularly if they have hypertension; such a risk is also present among normotensive nondipper patients with diabetes.²² The white-coat effect and placebo effect would not apply here because volunteers are free from the stress of a physician's presence during sleeping time. Through treatment with the mixed α- and β-blocker arotinolol, commonly used in Japan to manage hypertension, night-time SBP and DBP were significantly reduced only in nondipper patients;²³ however, reports of outcomes that support the potential of other types of antihypertensive agents in treatment of nondippers have been insufficient. To our knowledge, ours is the first study reporting that VPP and IPP cause a large decrease in early-morning blood pressure in nondipper patients. This finding suggests that VPP and IPP may have the potential to delay cardiovascular events in early morning.

In an earlier study, the prevalence of the nondipping profile was significantly higher in patients considered to be at high cardiovascular risk (42%) than in those with a medium (33%) or low (29%) risk.²⁴ It has also been suggested that endothelial function is impaired in hypertensive patients with a nondipper profile and endothelium-dependent vasodilation than dippers because of lower levels of nitric oxide release.²⁵ In treated hypertensive patients, cardiac structure was unaffected by dipping patterns.²⁶ In the present study, the trend observed here—that the food-derived ACE inhibitory peptides VPP and IPP were active in nondipper patients in the early morning—has not been reported with use of ACE inhibitory drugs. The reason that these 2 ACE inhibitory peptides were more effective in nondipper patients than dipper patients is not clear because the mechanism of nondipping is not well understood. Part of the explanation may be potent actions of VPP and IPP on vascular endothelial function.^27
–29 Another possibility is that unknown in vivo functions of VPP and IPP may be involved in their mode of action.

This study used L. helveticus–fermented milk containing VPP and IPP as a test product to determine whether treatment by these peptides would alter the circadian rhythm of blood pressure. Preparation of VPP and IPP from milk casein by an enzymatic treatment with Aspergillus oryzae has been reported.³⁰ The powdered peptides obtained by the methods described earlier are easily used in various kinds of foodstuff. Therefore, a casein hydrolysate prepared from a natural source would be a very promising ingredient for people with hypertension and those with normal daytime blood pressure but high night-time blood pressure. We recognize that this study is an uncontrolled and preliminary one. However, given the promising results, further research in this area is warranted.

Footnotes

Author Disclosure Statement

M.T.K., Y.N., N.Y., and K.Y. are employed as research scientists and headquarters office by Calpis Co., Ltd., Japan. No competing financial interests exist for T.I.

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