Casein Hydrolysate Containing the Antihypertensive Tripeptides Val-Pro-Pro and Ile-Pro-Pro Improves Vascular Endothelial Function Independent of Blood Pressure–Lowering Effects: Contribution of the Inhibitory Action of Angiotensin-Converting Enzyme

489 Hypertens Res Vol.30 (2007) No.6 p.489-496 Original Article Casein Hydrolysate Containing the Antihypertensive Tripeptides Val-Pro-Pro and Ile-Pro-Pro Improves Vascular Endothelial Function Independent of Blood Pressure–Lowering Effects: Contribution of the Inhibitory Action of Angiotensin-Converting Enzyme Tatsuhiko HIROTA1), Kohji OHKI1), Rikako KAWAGISHI2), Yoshitaka KAJIMOTO2), Seiichi MIZUNO1), Yasunori NAKAMURA1), and Masafumi KITAKAZE3) Accumulating evidence shows that deterioration of vascular endothelial function underlies the pathophysiology of cardiovascular diseases following lifestyle-related diseases. Both Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), which are tripeptides derived from proteolytic hydrolysate of milk casein, inhibit angiotensin-converting enzyme (ACE), suggesting that both VPP and IPP may improve vascular endothelial function, because many ACE inhibitors are known to improve endothelial function. We investigated the effects of ACE-inhibitory food component in humans with mild hypertension, since there has been no report on such effects. The study was conducted by the placebo-controlled, double-blind crossover method in 25 male subjects with mild hypertension. After casein hydrolysate containing both VPP and IPP were administered for 1 week, reactive hyperemia of the left upper forearm was measured using plethysmography as an index of vascular endothelial function. Since one subject dropped out, we analyzed the data of 24 subjects. The reactive hyperemia of the left upper forearm was produced by a 5 min occlusion using inflation of a cuff. The maximum blood flow during reactive hyperemia was 20.8 ± 6.7 mL/min/100 mL tissue in the placebo group, whereas it increased remarkably to 30.0 ± 10.4 mL/min/100 mL tissue in the group administered casein hydrolysate containing both VPP and IPP (p < 0.001). There was no change in systemic blood pressure, indicating that the improvement of the vascular endothelial function attributable to VPP and IPP is independent of hemodynamic changes. We conclude that casein hydrolysate containing VPP and IPP improves the vascular endothelial dysfunction in subjects with mild hypertension. The continuous intake of VPP and IPP could help to prevent cardiovascular diseases in hypertensive subjects. (Hypertens Res 2007; 30: 489–496) Key Words: reactive hyperemia, endothelial function, angiotensin-converting enzyme From the 1)R&D Center, Calpis Co., Ltd., Sagamihara, Japan; 2)Department of Clinical Trial and Research, Soiken Inc., Toyonaka, Japan; and 3)National Cardiovascular Center, Suita, Japan. Address for Reprints: Masafumi Kitakaze, M.D., Cardiovascular Division, National Cardiovascular Center, 5–7–1 Fujishirodai, Suita 565–8565, Japan. E-mail: Received September 19, 2006; Accepted in revised form January 12, 2007. 490 Hypertens Res Vol. 30, No. 6 (2007) 25 persons assessed for eligibility Introduction It has recently been recognized that lifestyle-related diseases such as hyperlipidemia, hypertension and diabetes mellitus precede cardiovascular diseases such as angina pectoris, myocardial infarction and cerebral vascular diseases. Lifestylerelated diseases, which are tightly linked to dietary habits and lifestyles, are on the rise worldwide and have contributed to an increase in cardiovascular events. The lifestyle-related diseases often overlap, and when the lifestyle-related diseases complicated, culminate in the progression of arteriosclerosis. The progression of arteriosclerosis consequently increases in the risk for cardiovascular diseases. There are many cellular explanations for the linkage between the lifestyle-related diseases and cardiovascular diseases. It is well known that the lifestyle-related diseases commonly deteriorate vascular endothelial function (1), one of the key factors for the progression of cardiovascular diseases. Indeed, vascular endothelial cells protect blood vessels and prevent arteriosclerosis by releasing various vasoactive substances that, in addition to their vasoactive properties, have the ability to inhibit monocyte adhesion and/or thrombus formation (2). Therefore, it is very important to protect or restore the endothelial function in patients with lifestyle-related diseases. Angiotensin-converting enzyme (ACE) inhibitors (ACEIs) have been known to decrease the incidence of cardiovascular diseases (3), while many ACEIs have been reported to improve vascular endothelial functions (4, 5). If common human foods could safely provide the ACE-inhibitory actions, their consumption could be a powerful strategy to prevent the incidence of cardiovascular diseases in patients with lifestyle-related diseases. Indeed, sardine peptide (6) derived from sardines, casein dodecapeptide, and dried bonito oligopeptide (7) have been demonstrated to be a source of natural food-derived ACEIs. Intriguingly, two tripeptides, Val-Pro-Pro (VPP) and Ile-ProPro (IPP), contained in Calpis fermented milk and proteolytic hydrolysate of milk casein also have the effects of ACEIs (8, 9), and we have reported that long-term intake of tablets, fermented milk, or other beverages containing both VPP and IPP resulted in blood pressure–lowering effects in subjects with mild hypertension (10), suggesting that VPP and IPP, with their ACE-inhibiting activities, can improve vascular endothelial function independent of their blood pressure–lowering effects, although to date there has been no report on the improvement of endothelial dysfunction by ACEIs derived from food components. Therefore, in the present study, we conducted a placebocontrolled crossover study to determine the effect of shortterm intake of casein hydrolysate containing both VPP and IPP on the endothelial function in subjects with mild hypertension. 1 retired for personal reasons 24 enrolled and randomized 12 assigned and received the test sample 12 assigned and received the placebo 12 assigned and received the placebo 12 assigned and received the test sample 24 analyzed Fig. 1. The trial profiles. Twelve patients received casein hydrolysate containing both Val-Pro-Pro (VPP) and Ile-ProPro (IPP) and 12 received the placebo in the first treatment period after enrollment. No significant difference was detected in the value of age, body height, body weight, body mass index, systolic blood pressure (SBP), diastolic blood pressure (DBP), pulse rate, blood glucose, HbA1c, total cholesterol and nitrate/nitrite (NOx), or number of cigarettes between the two groups (p> 0.2). Methods Subjects’ Characteristics and the Evaluation of Endothelial Function Twenty-five Japanese men with mild hypertension defined as a systolic blood pressure (SBP) of 140–159 mmHg or diastolic blood pressure (DBP) of 90–99 mmHg were enrolled in the study. Their ages ranged from 40 to 65 years (54.1±8.1 [mean±SD] years). Those who suffered from clinical disorders in liver function, or in the cardiovascular, respiratory, or endocrine systems were excluded from the study. (...truncated)