isoleucyl-prolyl-proline and valyl-prolyl-proline

The lactotripeptides isoleucine-proline-proline (IPP) and valine-proline-proline (VPP) have been shown to decrease systolic blood pressure (SBP) in several populations, but the size of the effect varies among studies. We performed a meta-analysis including all published studies to evaluate the SBP-lowering effect of IPP/VPP in Japanese subjects more comprehensively.. Eligible randomized controlled trials were searched for within four bibliographic databases, including two Japanese ones. Eighteen studies (including a total of 1194 subjects) were included in the meta-analysis. A random effect model using the restricted maximum likelihood (REML) estimator was used for the analysis. The analysis showed that consumption of IPP/VPP induced a significant reduction in SBP as compared with placebo in Japanese subjects, with an estimated effect of -5.63 mm Hg (95% CI, -6.87 to -4.39, P<0.0001) and no evidence of publication bias. A significant heterogeneity between series was evident, which could be explained by a significant influence of the baseline blood pressure status of the subjects, the effect of IPP/VPP on SBP being stronger in hypertensive subjects (-8.35 mm Hg, P<0.0001) than in non-hypertensive subjects (-3.42mm Hg, P<0.0001). Furthermore, the effect of IPP/VPP on SBP remained significant when limiting the analysis to series that tested the usual doses of IPP/VPP consumed daily (below 5 mg/d), with estimated effects of -6.01 mm Hg in the overall population and -3.32 mm Hg in non-hypertensive subjects.. Results from this meta-analysis show that IPP/VPP lactotripeptides can significantly reduce office SBP in Japanese subjects with or without overt hypertension, and for doses that can potentially be consumed as an everyday supplement. This suggests that these peptides could play a role in controlling blood pressure in Japanese subjects. The systematic review protocol was published on the PROSPERO register (CRD42014014322).

Topics: Adult; Angiotensin-Converting Enzyme Inhibitors; Blood Pressure; Humans; Hypertension; Japan; Middle Aged; Oligopeptides; Randomized Controlled Trials as Topic

Lactotripeptides (LTPs, including IPP and VPP) have held promise in the framework of lifestyle modification for prevention and control of hypertension - a cardiovascular risk factor, as LTPs are reported to have an inhibitory effect on angiotensin-converting enzyme. While the number of clinical trials to test the efficacy of LTP continues to increase, the results have been inconsistent, especially in the last few years. The purpose of the present meta-analysis is to precisely estimate the pooled mean effect of LTPs on conventional blood pressure (BP) generally and on 24-h ambulatory BP (ABP) particularly, as well as the change of BP in relation to baseline BP, race, and study design, to better reflect the evolving field.. In general analysis of 24 studies with 28 trials on 1919 human subjects, there are small reductions in both systolic BP (SBP) and diastolic BP (DBP) with the pooled mean effects of 1.66 (95% confidence interval (CI): -2.48 and -0.84) and 0.76 mmHg (-1.31 and -0.20) in response to LTP administration. In analysis of 24-h ABP response to LTP intervention, the reductions of SBP and DBP are 1.30 (-2.49 and -0.11) and 0.57 mmHg (-1.49 and 0.35). In subgroup analysis, the anti-hypertensive efficacy appears to be related to baseline BP, ethnic differences, treatment duration and double versus not double-blind design.. The present findings indicate that the BP-lowering effect of LTP is statistically significant, though small in magnitude. More clinical investigations (especially randomized double-blind trials with ABP) are warranted to determine the anti-hypertensive efficacy of LTP conclusively.

Topics: Antihypertensive Agents; Blood Pressure; Humans; Hypertension; Oligopeptides; Randomized Controlled Trials as Topic; Risk Factors

A meta-analysis of possible antihypertensive effects of small doses of bioactive tripeptides isoleucine-proline-proline and valine-proline-proline in commercial milk products or tablets was carried out. A random effects model was used on 19 randomized, placebo-controlled clinical intervention trials (published 1996-October 2010) consisting of about 1500 prehypertensive or mildly hypertensive subjects.The overall blood pressure lowering for systolic blood pressure was -4.0 mmHg (95% CI -5.9 to -2.1 mmHg, P < 0.001) and for diastolic blood pressure -1.9 mmHg (95% CI -3.1 to -0.8 mmHg, P < 0.001). However, a positive effect was not reported in all the studies. The results suggest that rather small daily doses of the lactotripeptides in different functional food products may offer a valuable option as a non-pharmacological treatment of prehypertension or mild hypertension as part of life-style advice.

Topics: Animals; Antihypertensive Agents; Blood Pressure; Confidence Intervals; Dietary Supplements; Humans; Hypertension; Milk Proteins; Oligopeptides; Randomized Controlled Trials as Topic

The milk-derived peptides isoleucine-proline-proline (IPP) and valine-proline- proline (VPP) have been shown to reduce systolic blood pressure (SBP). This decrease is convincingly shown in subjects of Asian origin, but less consistent results have been obtained in European populations.. A meta-analysis was conducted in accord with the Preferred Reporting Items for Systematic reviews and Meta-analyses (PRISMA) requirements, to assess the effect of IPP and VPP on SBP in Europeans, and to explore some determinants of this effect.. Ninety-one publications on the effect of IPP and VPP on SBP in Europeans were identified, and 14 trials with 15 sets of data (n = 1,306) met the inclusion criteria for the meta-analysis. A random-effects model (using the restricted maximum likelihood (REML) estimator) was used for the analysis. Although not all individual trials showed a statistically significant effect of IPP or VPP in reducing SBP, the combination of all data for the two peptides yielded a statistically significantly greater effect for IPP/VPP than for placebo. The decrease in SBP with IPP/VPP was 1.28mm Hg (95% CI, -2.09 to -0.48, P = 0.0017) and the decrease in diastolic BP (DBP) was 0.59mm Hg (95% CI, -1.18 to -0.01, P = 0.047). There was no evidence in the meta-analysis of any publication bias or of heterogeneity (P = 0.13). Among other features, a significant effect was seen for age, with each additional year of age reducing the effect on SBP by 0.09mm Hg. This might be related to isolated systolic hypertension, a condition often encountered in the elderly, who may be poorly responsive to first-line treatments for hypertension.. The peptides IPP and VPP are effective in moderately reducing SBP in European subjects, as is known for Asian populations. These two peptides could therefore have a role in controlling blood pressure (BP), a prospect that merits their further study.

Topics: Animals; Antihypertensive Agents; Asian People; Blood Pressure; Humans; Hypertension; Milk; Oligopeptides; Randomized Controlled Trials as Topic; White People

The oral assumption of lactotripeptides Valine-Proline-Proline (VPP) and Isoleucine-Proline-Proline (IPP) as nutraceuticals or functional foods is supposed to improve blood pressure (BP) control by angiotensin-converting enzyme-inhibition. However, data derived from clinical trials have reached conflicting conclusions. To perform a meta-analysis of placebo-controlled clinical trials evaluating the anti-hypertensive effect of lactotripeptides assumed as nutraceuticals or functional foods. Trials identified using a defined search strategy in PubMed were included in the meta-analysis, and their pooled effect was estimated with a random effects model, weighting for the inverse of the variance. Heterogeneity, publication bias, subgroup and meta-regression analyses were performed. A total of 18 trials have been identified, the clinical data of which have been clearly reported. Pooled effect of peptides was a reduction of -3.73 mm Hg (95% CI: -6.70, -1.76) for systolic blood pressure (SBP) and 1.97 mm Hg (95% CI: -3.85, -0.64) for diastolic blood pressure (DBP). The effect was more evident in Asian patients (SBP = -6.93 mm Hg (95% CI: -10.95, -2.94); DBP=-3.98 mm Hg(95% CI: -5.38, -2.44)) than in Caucasian ones (SBP=-1.17 mm Hg (95% CI: -2.82, 0.72); DBP = -0.52 mm Hg (95% CI: -1.39, 0.13)), and apparently not related to age, baseline BP values, dose of lactotripeptides assumed or length of the treatment. VPP and IPP lactotripeptides assumed as functional foods may significantly reduce SBP particularly in Asian subjects. The relevance of this findings in other ethnicities or associated with different dietary pattern should to be further investigated.

Topics: Adult; Asian People; Blood Pressure; Dietary Supplements; Female; Humans; Hypertension; Male; Middle Aged; Oligopeptides; Randomized Controlled Trials as Topic

Bioactive peptides derived from milk proteins are of particular interest to the food industry due to the potential functional and physiological roles that they demonstrate, particularly in relation to cardiovascular disease (CVD). By 2020 it is estimated that heart disease and stroke will become the leading cause of death and disability worldwide. Acute and chronic cardiovascular events may result from alterations in the activity of the renin-angiotensin aldosterone system and activation of the coagulation cascade and of platelets. Medications that inhibit angiotensin converting enzyme (ACE) are widely prescribed in the treatment and prevention of cardiovascular disease. ACE inhibitory peptides are of particular interest due to the presence of encrypted inhibitory peptide sequences. In particular, Ile-Pro-Pro and Val-Pro-Pro are fore runners in ACE inhibition, and have been incorporated into commercial products. Additionally, studies to identify additional novel peptides with similar bio-activity and the ability to withstand digestion during transit through the gastrointestinal tract are ongoing. The potential sources of such peptides in cheese and other dairy products are discussed. Challenges to the bio-availability of such peptides in the gastro intestinal tract are also reviewed. Activation of platelets and the coagulation cascade play a central role in the progression of cardiovascular disease. Platelets from such patients show spontaneous aggregation and an increased sensitivity to agonists which results in vascular damage and endothelial dysfunction associated with CVD. Peptide sequences exhibiting anti-thrombotic activity have been identified from fermented milk products. Studies on such peptides are reviewed and their effects on platelet function are discussed. Finally the ability of food derived peptides to decrease the formation of blood clots (thrombi) is reviewed. In conclusion, due to the widespread nature of cardiovascular disease, the identification of food derived compounds that exhibit a beneficial effect in such widespread areas of CVD regulation will have strong clinical potential. Due to the perception that food derived products have an acceptable risk profile they have the potential for widespread acceptance by the public. In this review, selected biological effects relating to CVD are discussed with a view to providing essential information to researchers.

Topics: Animals; Blood Platelets; Cardiovascular Diseases; Disease Models, Animal; Humans; Hypertension; Milk; Milk Proteins; Oligopeptides; Peptidyl-Dipeptidase A; Renin-Angiotensin System

The consumption of fermented milk to maintain good health, including the maintance of normal blood pressure, is an ancient tradition in a number of areas of the world (e.g., East Asia, France). Recent studies have suggested that fermented milk has a normotensive effect in hypertensive rats and humans, but no effect on blood pressure in normotensive rats and humans. Two tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP), have been identified as possessing significant angiotensin-converting enzyme inhibitory activity and are therefore believed to be the source of the normotensive effects. This document, the second of nine chapters, provides information on these two tripeptides, including physical/chemical properties, molecular weights, chemical structures, normal consumption in the diet, manufacturing information, regulatory approval in Japan, and Japanese consumption of food containing enhanced levels of VPP plus IPP. In addition, the results of studies in rats and humans conducted to evaluate the effect of these substances on blood pressure are presented. The research suggests that in adult normotensive volunteers, consumption of up to 7.92 mg of VPP and 4.52 mg IPP daily for 2 weeks causes neither clinical signs nor biologically meaningful effects on systolic or diastolic blood pressure, pulse rate, or clinical pathology (serum chemistry or hematology). However, when a similar study was performed using mildly and moderately hypertensive adults as subjects and they consumed 2.52 mg of VPP and 1.64 mg of IPP per day, a significant drop in systolic blood pressure was detected for a prolonged time interval. This chapter also introduces the issue of safety testing for these substances and describes the information to be found in the subsequent seven chapters.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Cultured Milk Products; Humans; Hypertension; Oligopeptides

Age-related declines in cognitive function and cerebral perfusion increase the risk of dementia. Although nutrition and exercise may be effective in reducing cognitive decline, the effect of lactotripeptide (LTP) on cerebral oxygenation and hemodynamics remains unclear.. The aim of this study was to investigate the effects of LTP ingestion on cerebral oxygenation, cognitive function, and vascular function in middle-aged and older adults with or without an exercise intervention.. We recruited 2 separate groups of participants, one with and one without an exercise intervention. Each group was then randomly assigned into a placebo group and an LTP group. The participants ingested a placebo or LTP every day. The exercise group performed aerobic exercises 4-6 d/wk. Before and after the 8-wk intervention, we measured oxygenated hemoglobin (oxy-Hb) concentration (oxy-Hb change) in the prefrontal cortex during the Stroop task (primary outcome), Stroop interference time, and carotid artery β-stiffness (both secondary outcomes).. Sixty-four participants completed the study. Changes in oxy-Hb signal in the prefrontal cortex were greater in the LTP group than in the placebo group under both the exercise and nonexercise conditions (P < 0.05). In addition, the magnitude of improvement in the oxy-Hb change in the left prefrontal cortex was correlated with Stroop interference (r = -0.39, P < 0.05) and carotid β-stiffness (r = -0.41, P < 0.05).. An 8-wk intake of LTP increased cerebral oxygenation in the prefrontal cortex region in middle-aged and older adults, with and without exercise. The intervention-induced improvements in brain neural activation were associated with cognitive and vascular function. This trial was registered at www.umin.ac.jp as UMIN000022313.

Topics: Aged; Animals; Carotid Arteries; Cognition; Cognitive Dysfunction; Executive Function; Exercise; Female; Hemodynamics; Humans; Male; Middle Aged; Milk; Oligopeptides; Oxyhemoglobins; Prefrontal Cortex; Respiratory Physiological Phenomena; Stroop Test; Vascular Stiffness

Lactotripeptides (LTPs) have a mild antihypertensive effect in hypertensive subjects. The main aim of our clinical trial was to test if LTPs could have some influence on blood pressure (BP) and related hemodynamic parameters in a sample of outpatients affected by metabolic syndrome.. A randomized, double-blind, placebo-controlled, crossover clinical trial was conducted in a group of 40 nonsmoking volunteers with metabolic syndrome. The treatment periods were 4 weeks long and were separated by a 4-week washout period. The dietary supplementation was made by daily administration of LTPs from casein, 10.2 mg/day, and compared with placebo.. During the LTP treatment, patients experienced a significant mean decrease in systolic BP (SBP; -3.4 ± 4.4 mmHg, P = 0.041), diastolic BP (DBP; -3.1 ± 3.2 mmHg, P = 0.049), and pulse wave velocity (PWV; -0.7 ± 0.3 m/sec, P = 0.001). After LTP treatment, delta SBP, DBP, and PP were all significantly improved (P < 0.01 for all) compared with placebo. PWV also improved significantly after LTP treatment with respect to the end of the treatment with placebo (-0.8 ± 0.4 vs. -0.1 ± 0.3 m/sec, P = 0.009). The square root of the ratio of peak:baseline pulse volume during hyperemia (√V2/V1) improved after LTP treatment only (1.2 ± 0.4 vs. 1.4 ± 0.5, P = 0.04). Through the evaluation of the hemodynamic parameters that were measured by the 24-hr ambulatory monitoring, we observed that SBP, MBP, and the percentage of time with SBP over the normal were significantly reduced only after the LTP treatment (P < 0.05). These parameters were also significantly improved when compared with the ones measured after the placebo treatment (P < 0.05).. In our trial, during LTP treatment, patients affected by metabolic syndrome experienced a mild but significant improvement in office and 24-hr BP, PWV, and endothelial function compared with placebo treatment.

Topics: Adult; Aged; Antihypertensive Agents; Blood Pressure; Cross-Over Studies; Double-Blind Method; Female; Humans; Hypertension; Male; Metabolic Syndrome; Middle Aged; Oligopeptides; Pulse Wave Analysis; Vascular Stiffness

Our aim was to evaluate the predictors of Isoleucine-Proline-Proline/Valine-Proline-Proline (IPP-VPP) lactotripeptides (LTPs) antihypertensive effect in the context of a short-term large double-blind randomized clinical trial involving 164 pharmacologically untreated subjects in primary prevention for cardiovascular disease. When compared with the baseline, office systolic blood pressure (SBP) (-3.42 mm Hg, P < .001) and diastolic blood pressure (DBP) (-2.35 mm Hg, P < .001) significantly decreased, in the LTP-treated patients only. No significant change in predictors during the study of ambulatory blood pressure measurement (ABPM) parameters was observed. A short-term supplementation with LTPs significantly improves the office SBP and DBP, especially in male subjects. The main predictor of LTP antihypertensive effect was the baseline BP.

Topics: Adult; Aged; Angiotensin-Converting Enzyme Inhibitors; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Cardiovascular Diseases; Caseins; Double-Blind Method; Female; Humans; Hypertension; Male; Middle Aged; Office Visits; Oligopeptides; Predictive Value of Tests; Time Factors; Treatment Outcome

Lactobacillus helveticus LBK-16H-fermented milk products containing tripeptides isoleucine-proline-proline and valine-proline-proline lower blood pressure in hypertensive subjects using office and home blood pressure registration. The present study was aimed to evaluate the effects of two doses of these lactotripeptides on 24-h ambulatory blood pressure and lipidomics profiles in mildly hypertensive subjects.. In a randomized, double-blind, placebo-controlled parallel group study, 89 mildly hypertensive subjects ingested, after a 1-month run-in period, a fermented milk drink with 5 mg per day of lactotripeptides during 3 months, and a milk drink with 50 mg per day of lactotripeptides for the following 3 months, or a placebo milk drink without lactotripeptides. Ambulatory blood pressure (24 h) was recorded at baseline and at the end of the intervention periods. Lipidomics profiles were characterized before and after the 6-month intervention.. After the second intervention period (50 mg per day of lactotripeptides), systolic and diastolic 24-h blood pressures decreased significantly in the peptide, but not in the placebo group. However, the treatment effects -2.6 mm Hg (95% confidence interval (CI): -5.7 to 0.4) in systolic and -1.3 mm Hg (95% CI: -3.4 to 0.8) in diastolic blood pressure did not reach statistic significance. Ingestion of 5 mg per day of lactotripeptides for 3 months did not lower blood pressure. The peptide group was dominated by decrease in multiple phospholipids (PL).. Ingestion of fermented milk with daily dose of 50 mg of lactotripeptides appears to lower elevated blood pressure slightly from the baseline, but not significantly compared with the placebo group and to induce significant decreases in multiple PL.

Topics: Adult; Antihypertensive Agents; Blood Pressure; Cardiovascular Diseases; Cultured Milk Products; Double-Blind Method; Female; Humans; Hypertension; Lactobacillus helveticus; Male; Middle Aged; Oligopeptides; Phospholipids; Risk Factors

A double-blind, placebo-controlled, randomized clinical trial was conducted to evaluate the effects of ingesting an excess of tablets containing casein hydrolysate, incorporating angiotensin I-converting enzyme (ACE) inhibitory peptides such as Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), in subjects with blood pressure ranging from normal to mild hypertension. A total of 48 subjects were given either 5 times more than the effective amount of casein hydrolysate or a placebo in tablet form for 4 weeks. In the active group, systolic blood pressure (SBP) decreased significantly as compared with the placebo group. In stratified analysis, however, this antihypertensive effect was not found in normotensive subjects. In addition, neither an acute or nor an excessive reduction in blood pressure nor clinically important adverse events were observed in this study. These findings suggest that intake of a 5-fold excess of tablets containing casein hydrolysate can lead to a mild improvement in hypertension without side effects.

Topics: Angiotensin-Converting Enzyme Inhibitors; Antihypertensive Agents; Blood Pressure; Body Mass Index; Body Weight; Caseins; Double-Blind Method; Female; Humans; Hypertension; Japan; Male; Middle Aged; Oligopeptides; Peptidyl-Dipeptidase A; Placebos; Tablets

Casein-derived tripeptides isoleucine-proline-proline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro) lower blood pressure (BP) in long-term clinical studies. Their acute effects on BP and vascular function, important for daily dosing scheme, were studied in a placebo-controlled double-blind crossover study using a single oral dose of a fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro as well as plant sterols. Twenty-five subjects with untreated mild hypertension received in random order 250 g of study product (25 mg peptides and 2 g plant sterols) or placebo. Ambulatory BP was monitored for 8 h post-dose and arterial stiffness measured by pulse wave analysis at 2, 4, and 8 h. Blood and urine samples were analyzed for markers of the renin-angiotensin system (RAS) and endothelial function. Baseline adjusted treatment effect for systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial BP was -2.1 mmHg (95% CI: -4.1 to -0.1, p = 0.045), -1.6 mmHg (95% CI: -3.1 to -0.1, p = 0.03), and -1,9 mmHg (95% CI: -3-3 to -0.4, p = 0.0093), respectively, in favor of the active treatment for 8 h post- dose. No significant differences between the treatments were seen in brachial or aortic augmentation index, pulse wave velocity, or markers of RAS. Urinary excretion of cGMP, the second messenger of endothelial nitric oxide, was higher in the active group vs. placebo (p = 0.01). The results indicate that a single dose of a fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro and plant sterols acutely lowers brachial SBP and DBP in mildly hypertensive subjects.

Topics: Administration, Oral; Animals; Antihypertensive Agents; Blood Pressure; Cross-Over Studies; Cultured Milk Products; Cyclic GMP; Dose-Response Relationship, Drug; Double-Blind Method; Female; Humans; Hypertension; Male; Middle Aged; Oligopeptides; Phytosterols; Renin-Angiotensin System; Treatment Outcome

This trial evaluated the effects of casein hydrolysate containing milk-derived peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), on central blood pressure and arterial stiffness.. A randomized, double-blind, placebo-controlled trial was conducted in 70 Japanese subjects aged 50-69 years with untreated stage-I hypertension. They were randomly assigned to two groups, which received either placebo tablets or active tablets containing 3.4 mg of VPP and IPP. At the beginning and end of the 8-week intervention, hemodynamic parameters, including central blood pressure and brachial-ankle pulse wave velocity (baPWV), a marker of arterial stiffness, were measured.. A significant difference in changes in central systolic blood pressure between the groups was observed (active: -11.0±11.0 vs placebo: -4.5±9.6 mmHg, P<0.01). In the active group, reductions in baPWV (-73.9±130.0 vs -8.4±137.1 cm/s, P<0.05), brachial SBP (-10.5±11.5 vs -3.9±9.6 mmHg, P<0.05), and radial mean blood pressure (-7.3±8.9 vs -2.0±7.4 mmHg, P<0.01) were significantly greater as compared with the placebo group.. Casein hydrolysate containing VPP and IPP improves central SBP and baPWV in hypertensive subjects, which suggests VPP and IPP might have beneficial effects on arterial properties.

Topics: Aged; Angiotensin-Converting Enzyme Inhibitors; Ankle Brachial Index; Antihypertensive Agents; Blood Pressure; Caseins; Double-Blind Method; Female; Humans; Hypertension; Male; Middle Aged; Oligopeptides; Vascular Stiffness

Contrasting data partially support a certain antihypertensive efficacy of lactotripeptides (LTPs) derived from enzymatic treatment of casein hydrolysate. Our aim was to evaluate this effect on a large number of hemodynamic parameters. We conducted a prospective double-blind randomized clinical trial, which included 52 patients affected by high-normal blood pressure (BP) or first-degree hypertension. We investigated the effect of a 6-week treatment with the LTPs isoleucine-proline-proline and valine-proline-proline at 3 mg per day, assumed to be functional food, on office BP, 24-h ambulatory BP monitoring (ABPM) values, stress-induced BP increase and cardiac output-related parameters. In the LTP-treated subjects, we observed a significant reduction in office systolic BP (SBP; -5±8 mm Hg, P=0.013) and a significant improvement in pulse wave velocity (PWV; -0.66±0.81 m s(-1), P=0.001; an instrumental biomarker of vascular rigidity). No effect on 24-h ABPM parameters and BP reaction to stress was observed from treatment with the combined LTPs. LTPs, but not placebo, were associated with a mild but significant change in the stroke volume (SV), SV index (markers of cardiac flow), the acceleration index (ACI) and velocity index (VI) (markers of cardiac contractility). No effect was observed on parameters related to fluid dynamics or vascular resistance. LTPs positively influenced the office SBP, PWV, SV, SV index, ACI and VI in patients with high-normal BP or first-degree hypertension.

Topics: Adult; Aged; Blood Flow Velocity; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Cardiac Output; Caseins; Double-Blind Method; Female; Heart Rate; Humans; Hypertension; Male; Middle Aged; Oligopeptides; Pulsatile Flow; Stress, Physiological

Peripheral conduit artery endothelium-dependent dilatation decreases with aging in humans. Lactotripeptides (LTPs) and regular exercise can improve endothelium-dependent dilatation, but combining these lifestyle modifications may be more effective than either treatment alone. We conducted a randomized, place-controlled trial with four different intervention arms.. A total of 43 postmenopausal women (50-65 years old) were randomly divided into placebo, LTP, exercise and placebo (Ex+placebo), or exercise and LTP (Ex+LTP) groups. LTP or placebo was administered orally for 8 weeks. The exercise groups completed an 8-week moderate aerobic exercise (walking or cycling) intervention.. There were no statistically significant differences in baseline flow-mediated dilatation (FMD) and most other key dependent variables among the groups. FMD significantly increased in the LTP, Ex+placebo, and Ex+LTP groups whereas no such changes were observed in the placebo control group. The magnitude of increases in FMD was significantly greater in the Ex+LTP group than other intervention groups.. We concluded that LTP ingestion combined with regular aerobic exercise improves endothelium-dependent dilatation to a greater extent than monotherapy with either intervention alone in postmenopausal women.

Topics: Aged; Blood Flow Velocity; Blood Pressure; Cardiovascular Diseases; Combined Modality Therapy; Dietary Supplements; Dose-Response Relationship, Drug; Endothelium-Dependent Relaxing Factors; Endothelium, Vascular; Exercise; Female; Humans; Middle Aged; Oligopeptides; Postmenopause; Vasodilation

The milk casein-derived biologically active tripeptides, isoleucyl-prolyl-proline (Ile-Pro-Pro) and valyl-prolyl-proline (Val-Pro-Pro), have documented antihypertensive effect probably related to reduced angiotensin formation. It has been suggested that these tripeptides may reduce arterial stiffness and improve endothelial function. Our aim was to evaluate whether the milk-based drink containing Ile-Pro-Pro and Val-Pro-Pro influence arterial stiffness, measured as augmentation index (AIx), and endothelial function in man.. In a double-blind parallel group intervention study, 89 hypertensive subjects received daily peptide milk containing a low dose of tripeptides (5 mg/day) for 12 weeks and a high dose (50 mg/day) for the following 12 weeks, or a placebo milk drink to titrate the dose-response effect. Arterial stiffness was assessed by pulse wave analysis at the beginning and end of each intervention period. Endothelial function was tested by examining pulse wave reflection response to sublingual nitroglycerin and salbutamol inhalation. Blood pressure was measured by using office and 24-h ambulatory blood pressure measurement.. At the end of the second intervention period, AIx decreased significantly in the peptide group compared with the placebo group (peptide group -1.53% (95% confidence interval (CI) -2.95 to -0.12), placebo group 1.20% (95% CI 0.09-2.32), P=0.013). No change in endothelial function index was observed (peptide group 0.02 (95% CI -0.06 to 0.08), placebo group 0.04 (95% CI -0.04 to 0.12), P=0.85). There were no statistically significant differences between the effects of the peptide and placebo treatment on office and 24-h ambulatory blood pressure.. Long-term treatment with Lactobacillus helveticus-fermented milk containing bioactive peptides reduces arterial stiffness expressed as AIx in hypertensive subjects.

Topics: Adult; Animals; Antihypertensive Agents; Blood Pressure; Caseins; Double-Blind Method; Endothelium, Vascular; Female; Fermentation; Food Microbiology; Humans; Hypertension; Lactobacillus helveticus; Male; Middle Aged; Milk; Oligopeptides; Radial Artery

Nearly 70 million Americans have hypertension, and approximately an equal number have prehypertension. The prevalence of both disorders increases with advancing age and obesity. Many at-risk individuals do not have controlled blood pressure (BP). Lifestyle modification for most persons is the first step in a plan to control these conditions. Non-drug treatments offer an appeal to many patients with modest BP elevation. The authors recently evaluated BP response using 24-hour ambulatory BP monitoring and office BP monitoring of lactotripeptides dosed twice daily in 91 previously treated and treatment-naive patients with stage 1 and stage 2 hypertension. In this population, daytime systolic BP, the primary efficacy end point, significantly decreased (-3.6 mm Hg; P=.013), while placebo did not affect systolic BP (0 mm Hg; P=not significant). Treatment-naive patients exhibited a more robust drop in their daytime systolic BP (-7.6 mm Hg; P=.005) compared with placebo (-3.6 mm Hg; P=not significant). Lactotripeptides may be an effective agent in the management of low-risk and low-grade hypertension and prehypertension.

Topics: Adult; Aged; Antihypertensive Agents; Blood Pressure Monitoring, Ambulatory; Dose-Response Relationship, Drug; Double-Blind Method; Drug Administration Schedule; Drug Combinations; Female; Humans; Hypertension; Male; Middle Aged; Oligopeptides; Prospective Studies

It is well known that the sour milk containing lactotripeptides has a blood pressure lowering effect. The aim of this study was to evaluate the blood pressure (BP) lowering effect of lactotripeptides by monitoring home blood pressure, 24-h ambulatory measurements (ABPM), and daily urinary salt excretion. A total of 30 volunteers were given 200 ml of sour milk twice a day for 8 weeks after a 1-week run-in period. This preparation contained the lactotripeptides valine-proline-proline 2.66 mg and isoleucine-proline-proline 1.38 mg. The study participants had daily measurements of urinary salt excretion determined by an electric salt sensor and home blood pressure for each week during the run-in period, before the 4-and 8-week time points. 24-h ABPM was measured at the end of each week. Mean systolic blood pressure (SBP) during night sleep including base BP at 4 and 8 weeks were significantly lower than baseline values. Mean SBP and diastolic blood pressure (DBP) during night sleep of the 22 participants who belonged to the criteria of hypertension by 24-h ABPM was significantly decreased at 4 and 8 weeks. The change in 24-h mean SBP significantly correlated with mean urinary salt excretion over the three measurement periods. The 22 hypertensive subjects without taking lactotripeptides did not show significant change of blood pressure during 24 hours at 4 and 8 weeks. Our study confirmed the BP lowering effect of lactotripeptides during night-time sleep and showed that a lower intake of salt may increase the BP lowering effect of lactotripeptides through 24 hours in hypertensive subjects.

Topics: Adult; Aged; Algorithms; Blood Pressure Monitoring, Ambulatory; Circadian Rhythm; Cultured Milk Products; Female; Humans; Hypertension; Male; Middle Aged; Oligopeptides; Sodium; Sodium Chloride, Dietary; Systole; Treatment Outcome

Contrasting data partially support a certain antihypertensive efficacy of lactotripeptides derived from enzymatic treatment of casein hydrolysate. We carried out a randomized, double-blind, crossover clinical study to investigate the antihypertensive efficacy of a short-term treatment with lactotripeptides in Mediterranean subjects with normal or high-normal blood pressure (BP). We consecutively enrolled 55 untreated subjects (men:women = 30:25), 40.3 ± 9.8 years old, with normal or high-normal BP. After 4 weeks of dietary standardization, they were allocated to treatment with a fruit juice containing 3 mg of added Ile-Pro-Pro/Val-Pro-Pro lactotripeptides or with placebo for 4 weeks. After a 4-week washout period, they were then assigned to the alternative treatment for a further period of 4 weeks. Overall, no significant difference has been observed in office BP comparing baseline data with those posttreatment. Repeating the analysis by basal BP level, a mild but significant reduction in systolic BP (-1.7 ± 2.3 mm Hg; t = 3.5, P = .002) has been observed only in subjects with high-normal BP after treatment with lactotripeptides. With regard to 24-hour BP measurement, after lactotripeptide treatment only, the subjects experienced a significant reduction in diurnal diastolic BP (-1.6 ± 5.4 mm Hg; P = .042), diurnal mean BP (-2.1 ± 5.9 mm Hg; P = .19), and 24-hour (-5.4 ± 14.2 mm Hg; P = .011) and diurnal (-7.1 ± 19.2%; P = .014) diastolic BP value measurements relative to normal values. No modification has been observed in relation to plasma renin activity and aldosteronemia. In conclusion, diurnal diastolic BP is significantly reduced by lactrotripeptide supplementation in untreated Mediterranean subjects with normal or high-normal BP. Office systolic BP is reduced only in subjects with high-normal BP.

Topics: Adult; Aged; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Cardiovascular Diseases; Caseins; Circadian Rhythm; Cross-Over Studies; Dietary Supplements; Double-Blind Method; Female; Hemodynamics; Humans; Hypertension; Male; Mediterranean Region; Middle Aged; Oligopeptides; Severity of Illness Index; Young Adult

Dietary factors directly influence blood pressure (BP). The lactotripeptides (LTPs) IPP (isoleucine-proline-proline) and VPP (valine-proline-proline), formed by hydrolyzing dairy proteins, and potassium, a mineral mainly found in fruit, vegetables, and dairy products, are extensively studied for their BP-lowering effect. The efficacy of LTPs seems modest in whites compared with that in Asians.. The objective was to study the effects of enzymatically produced LTPs alone or in combination with potassium on ambulatory BP in whites.. Two multicenter, placebo-controlled, randomized, crossover studies were conducted; each consisted of two 4-wk intervention periods separated by a 4-wk washout period. In study 1, 69 subjects received 200 g/d of a dairy drink with 5.8 mg IPP and 4.4 mg VPP or placebo. In study 2, 93 subjects received 100 g/d of a dairy drink with 2.7 mg IPP, 1.9 mg VPP, and 350 mg added K or placebo. The subjects were randomly assigned according to their daytime ambulatory BP.. Mean 24-h systolic and diastolic BP (baseline values-study 1: 137.1/81.6 mm Hg; study 2: 139.2/80.9 mm Hg) remained similar with no significant differences between treatments in either study (P > 0.10). Office BP decreased over the course of both studies (systolic BP > 5 mm Hg), but differences between interventions were not significant (P > 0.10). In both studies, nighttime BP dipped during all treatments (> or =15%) but was statistically more significant with placebo (P < 0.05). Sodium excretion increased significantly after consumption of LTPs and potassium compared with after placebo intervention (P = 0.01), but not after consumption of LTPs alone.. The data do not support a BP-lowering effect of LTPs in whites.

Topics: Angiotensin-Converting Enzyme Inhibitors; Blood Pressure; Circadian Rhythm; Cross-Over Studies; Cultured Milk Products; Diastole; Female; Humans; Male; Oligopeptides; Potassium; Systole; White People

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.

Topics: Aged; Beverages; Blood Pressure Determination; Blood Pressure Monitoring, Ambulatory; Caseins; Dose-Response Relationship, Drug; Double-Blind Method; Female; Humans; Hypertension; Male; Middle Aged; Office Visits; Oligopeptides; Powders; Reproducibility of Results; Yogurt

Central arterial compliance plays an important role in the functional abilities of the vasculature. Two active tripeptides, valine-proline-proline and isoleucine-proline-proline, were isolated from sour milk and were referred to as lactotripeptides (LTP). Because LTP appears to act as an angiotensin-converting enzyme inhibitor, it is plausible to hypothesize that LTP improves arterial compliance. We determined the effects of LTP ingestion alone or in combination with regular aerobic exercise on arterial compliance. A total of 55 postmenopausal women (50-65 yr old) were randomly divided into four groups: placebo, LTP, exercise and placebo (Ex + placebo), or exercise and LTP (Ex + LTP). LTP or placebo was administered orally for 8 wk. The exercise groups completed an 8-wk moderate aerobic exercise intervention. There were no differences in baseline arterial compliance and most other key dependent variables among the groups. Carotid arterial compliance increased significantly in the LTP (0.93 + or - 0.07 vs. 0.99 + or - 0.08 mm(2)/mmHg x 10(-1)), Ex + placebo (0.92 + or - 0.04 vs. 1.00 + or - 0.05 mm(2)/mmHg x 10(-1)), and Ex + LTP groups (0.86 + or - 0.06 vs. 1.00 + or - 0.06 mm(2)/mmHg x 10(-1)), whereas no such changes were observed in the placebo control group (0.86 + or - 0.06 vs. 0.85 + or - 0.07 mm(2)/mmHg x 10(-1)). The magnitude of increases in carotid arterial compliance was significantly greater in the Ex + LTP group (19 + or - 4%) than in other groups. The improvements in arterial compliance with LTP were associated with the corresponding reductions in arterial blood pressure and plasma angiotensin II concentrations. We concluded that LTP ingestion improves carotid arterial compliance and that the combination of LTP ingestion and regular exercise is additive and synergistic in improving arterial compliance in postmenopausal women.

Topics: Administration, Oral; Aged; Angiotensin II; Blood Pressure; Brachial Artery; Carotid Arteries; Compliance; Cultured Milk Products; Dietary Supplements; Exercise; Female; Humans; Middle Aged; Oligopeptides; Oxygen Consumption; Postmenopause; Tablets; Ultrasonography

Pharmaceutical angiotensin-converting enzyme (ACE) inhibitors have been shown to reduce arterial stiffness; the possible effect of food-derived putative ACE inhibitory peptides on this degenerative process, however, has not been reported. In the present study, casein hydrolysate containing the lactotripeptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), which has been found to have an antihypertensive effect in a number of clinical studies, was investigated for its ability to improve hemodynamic parameters, including central systolic blood pressure (cSBP), in hypertensive subjects. Twelve hypertensive subjects who were not on prescribed medication were monitored for various hemodynamic parameters, including brachial blood pressure (peripheral blood pressure), cSBP, and augmentation index (AI), at the start and then after 3, 6, and 9 weeks of a daily treatment comprising four tablets containing VPP and IPP. Compared with basal levels, treatment with casein hydrolysate for 6 and/or 9 weeks showed a significant reduction in peripheral systolic and diastolic blood pressure, AI, and cSBP, but not in heart rate or pulse pressure. cSBP showed a reduction sooner and greater (-21.8 mm Hg) than did brachial systolic blood pressure (-16.4 mm Hg) during the 9-week treatment. Although small and not placebo-controlled, this study suggests that continuous intake of VPP and IPP might have the potential to improve arterial stiffness as well as cSBP and peripheral brachial blood pressure.

Topics: Adult; Blood Pressure; Caseins; Female; Hemodynamics; Humans; Hypertension; Male; Middle Aged; Oligopeptides

Several placebo-controlled clinical studies suggest that products containing isoleucyl-prolyl-proline and valyl-prolyl-proline are able to lower blood pressure without adverse effects. The most efficient way of producing high concentrations of these lactotripeptides (LTPs) is enzymatic hydrolysis of dairy protein (casein) with the use of a mixture of several enzymes derived from the nongenetically modified organism Aspergillus oryzae, including proteases and peptidases. To date, no large studies of the blood pressure-lowering properties of enzymatically produced LTP (ELTP) powder in European populations have been published.. This study was performed to evaluate the hypothesis that consumption of ELTP in a yogurt beverage for 8 wk significantly lowers blood pressure.. In this multicenter, double-blind, parallel, placebo-controlled trial, office blood pressure was evaluated in 275 Dutch hypertensive subjects. Blood pressures and body weight were measured on several days at baseline and at weeks 4 and 8 of the intervention between 2.5 and 3 h after intake of the test product. Twenty-four-h urine samples were collected at baseline and at the end of the intervention for urinalysis of sodium, potassium, creatinine, and microalbumin excretion.. The results showed that 10.2 mg ELTP/d does not lead to a reduction in systolic blood pressure (P = 0.66) or diastolic blood pressure (P = 0.72) compared with placebo.. This study showed no effect of an ELTP-enriched yogurt beverage on blood pressure in hypertensive subjects in a fairly large study.

Topics: Adult; Aged; Angiotensin-Converting Enzyme Inhibitors; Blood Pressure; Cultured Milk Products; Diastole; Double-Blind Method; Female; Humans; Hypertension; Male; Middle Aged; Netherlands; Oligopeptides; Peptides; Systole; Treatment Failure; Urinalysis; Yogurt

Milk-derived peptides with ACE-inhibiting properties may have antihypertensive effects in humans. We conducted a randomized double-blind placebo-controlled trial to examine the blood pressure lowering potential of 2 ACE-inhibiting lactotripeptides, ie, Isoleucine-Proline-Proline and Valine-Proline-Proline. We included 135 Dutch subjects with elevated systolic blood pressure who were otherwise healthy and who received no current antihypertensive treatment. After a 2-week run-in period on placebo, subjects randomly received a daily dose of 200 mL dairy drink with 14 mg lactotripeptides obtained by concentrating fermented milk, enzymatic hydrolysis, or chemical synthesis, or placebo for 8 weeks, followed by a 2-week wash-out. The primary outcome was 8-week change in office systolic blood pressure. Secondary outcomes were change in diastolic blood pressure, home blood pressure, 24-hour ambulatory blood pressure, plasma ACE-activity, and plasma angiotensin II. Blood pressure at baseline was on average 142/84 mm Hg. Lactotripeptides did not significantly change systolic blood pressure (P=0.46) or diastolic blood pressure (P=0.31) compared with placebo. The mean difference (95%-CI) in systolic blood pressure response between treatment and placebo was 2.8 mm Hg (-2.6;8.2) for concentrated fermented milk lactotripeptides, -0.5 mm Hg (-6.0;5.0) for enzymatic lactotripeptides, and 1.6 mm Hg (-3.9;6.9) for synthetic lactotripeptides. Treatment neither had a significant effect on secondary outcome measures. In conclusion, the present study does not support the hypothesis of a blood pressure lowering effect of the lactotripeptides Isoleucine-Proline-Proline and Valine-Proline-Proline.

Topics: Adult; Aged; Angiotensin-Converting Enzyme Inhibitors; Blood Pressure; Cultured Milk Products; Diastole; Double-Blind Method; Female; Humans; Male; Middle Aged; Oligopeptides; Patient Compliance; Peptides; Systole

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.

Topics: Adult; Aged; Angiotensin-Converting Enzyme Inhibitors; Biomarkers; Blood Pressure; Caseins; Cross-Over Studies; Endothelium, Vascular; Forearm; Humans; Hypertension; Male; Middle Aged; Oligopeptides

Two tripeptides (Val-Pro-Pro and Ile-Pro-Pro) that have inhibitory activities for angiotensin I-converting enzyme are produced in milk fermented with Lactobacillus helveticus. In this study we evaluated the effect and safety of powdered fermented milk with L. helveticus CM4 on subjects with high-normal blood pressure or mild hypertension.. A randomized, placebo-controlled, double-blind study was conducted using 40 subjects with high-normal blood pressure (HN group) and 40 subjects with mild hypertension (MH group). Each subject ingested 6 test tablets (12 g) containing powdered fermented milk with L. helveticus CM4 daily for 4 weeks (test group) or the same amount of placebo tablets for 4 weeks (placebo group).. During treatment, the decrease in systolic blood pressure (SBP) and diastolic blood pressure (DBP) in the test group tended to be greater than in the placebo group for both blood pressure groups. At the end of treatment (week 4), a significant decrease in DBP in the HN group was observed (i.e. 5.0 mm Hg (0.1, 9.9; p = 0.04) compared with the placebo group). There was no significant change in SBP (3.2 mm Hg (95% CI -2.6, 8.9; p = 0.27). In the MH group, SBP decreased by 11.2 mm Hg (4.0, 18.4; p = 0.003) and there was a statistically non-significant decrease in DBP of 6.5 mm Hg (-0.1, 13.0; p = 0.055) compared with the placebo group. No marked changes were observed in other indexes, including pulse rate, body weight and blood serum variables, and no adverse effects attributed to the treatment was found in each group.. Daily ingestion of the tablets containing powdered fermented milk with L. helveticus CM4 in subjects with high-normal blood pressure or mild hypertension reduces elevated blood pressure without any adverse effects.

Topics: Animals; Antihypertensive Agents; Blood Pressure; Consumer Product Safety; Double-Blind Method; Female; Fermentation; Humans; Hypertension; Lactobacillus; Male; Middle Aged; Milk; Oligopeptides; Probiotics

We describe a clinical trial to study the efficacy of a casein hydrolysate, prepared using an Aspergillus oryzae protease, containing the major angiotensin-I-converting enzyme inhibitory peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) in a single-blind, placebo-controlled study. A total of 131 volunteers with high-normal blood pressure and mild hypertension were randomly divided into four groups (n 32 or 33 in each group). 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 systolic blood pressure was observed at 6 weeks in the active group receiving 1.8 mg (P<0.01) VPP and IPP; in the active groups receiving either 2.5 mg or 3.6 mg, systolic blood pressure was decreased at both 3 weeks (P<0.05 and P<0.05) and 6 weeks (P<0.001 and P<0.0001) compared with systolic blood pressure measured before treatment. Changes in the systolic blood pressure 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 systolic blood pressure between the placebo group and the VPP and IPP group receiving 3.6 mg was observed (P<0.001) by two-way ANOVA. The antihypertensive effect was greater in mildly hypertensive subjects (n 20 or 21 in each group) than in any of the other subjects. No significant change of diastolic blood pressure was observed for all the test groups, and no differences in diastolic blood pressure in the test sample groups compared with the placebo group were observed during the test period.

Topics: Administration, Oral; Adult; Analysis of Variance; Angiotensin-Converting Enzyme Inhibitors; Antihypertensive Agents; Aspergillus oryzae; Blood Pressure; Body Mass Index; Body Weight; Caseins; Dietary Supplements; Female; Humans; Hypertension; Male; Oligopeptides; Single-Blind Method

The present study was carried out to evaluate the blood pressure (BP)-lowering effect and the safety aspects of Lactobacillus helveticus LBK-16H fermented milk with high tripeptide doses on hypertensive subjects using 24-h ambulatory measurements (ABPM).. In a randomized, double blinded placebo-controlled parallel group study, 94 hypertensive patients not receiving any drug treatment were given 150 mL twice daily of either L. helveticus LBK-16H fermented milk with a high concentration of tripeptides (Ile-Pro-Pro 7.5 mg/100 g and Val-Pro-Pro 10 mg/100 g) or a control product, for 10 weeks after a 4-week run-in period. Twenty-four-hour ABPM were taken at the beginning and at the end of the intervention period. The average baseline systolic and diastolic BP values were 132.6 +/- 9.9/83.0 +/- 8.0 mm Hg in the L. helveticus group and 130.3 +/- 9.6 /80.2 +/- 7.0 mm Hg in the control group.. There was a mean difference of -4.1 +/- 0.9 mm Hg in systolic (P = .001) and a -1.8 +/- 0.7 mm Hg in diastolic BP (P = .048) between the L. helveticus group and the control group. There was no difference in the sum of the adverse events (P = .820).. Lactobacillus helveticus LBK-16H fermented milk containing bioactive peptides, in daily use, does have a BP-lowering effect in hypertensive subjects and is thus a potential for the dietary treatment of hypertension.

Topics: Analysis of Variance; Animals; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Circadian Rhythm; Double-Blind Method; Female; Fermentation; Humans; Hypertension; Lactobacillus helveticus; Male; Milk; Milk Proteins; Office Visits; Oligopeptides

Angiotensin-converting enzyme (ACE) is important in the regulation of blood pressure (BP). Two tripeptides that inhibit ACE, isoleucyl-prolyl-proline (Ile-Pro-Pro) and valyl-prolyl-proline (Val-Pro-Pro), have been isolated from certain sour milks. The aim of the study reported was to evaluate the effect on BP in subjects with mild hypertension of a new sour milk containing tripeptides. The initial number of subjects was 60 (36 men, 24 women). Among the criteria for inclusion in the study were systolic BP (SBP) between 140 and 180 mmHg and/or diastolic BP (DPB) between 90 and 110 mmHg, without antihypertensive drug therapy. There were two study periods with a washout period between. All subjects were given 1.5 dl per day of a placebo (regular sour milk) or of the active product, a milk that had been fermented with Lactobacillus helveticus bacteria and contained 2.4-2.7 mg of Ile-Pro-Pro and 2.4-2.7 mg of Val-Pro-Pro per 1.5 dl. In the first phase, SBP fell 16 mmHg from baseline in the active group, 2 mmHg more than in the placebo group (P=0.0668) and no difference in DBP (P=0.92). There was a statistically significant downward trend both in SBP and DBP (P=0.0001). During the second phase, SBP fell 11 mmHg in the active group (P=0.008). The reduction in SBP was significantly larger in active than placebo group (P=0.012). In the crossover analysis combining both phases, SBP fell on average 2.6+/-15.9 mmHg more on the active product compared with the placebo product, but this difference was not statistically significant (P=0.3111). The difference in DBP, 1.0+/-8.3 mmHg between the two test products was not significant either (P=0.4431). In conclusion, the ingestion of sour milk fermented by L. helveticus bacteria and that containing ACE inhibitory tripeptides seems to lower BP modestly.

Topics: Analysis of Variance; Cross-Over Studies; Female; Fermentation; Finland; Humans; Hypertension; Lactobacillus; Male; Middle Aged; Milk Proteins; Oligopeptides

Angiotensin II (Ang II), a vasoactive factor in the renin-angiotensin-aldosterone system (RAAS), can regulate vasoconstriction and promote multiple vascular diseases. In this study, the effects of potent antihypertensive peptide Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) on the proliferation and migration of vascular smooth muscle cells (VSMCs) by extracellular vesicles (EVs) from vascular endothelial cells (VECs) were studied using a cell co-culture model. The VEC-derived EVs were isolated, characterized, and investigated. The present study demonstrated that the EVs from Ang II-induced VECs could promote proliferation, migration, and inflammatory factors (IL-6 increased to 40.75 ± 4.33 pg/mL and IL-1β increased to 28.62 ± 5.42 pg/mL) generation of VSMCs, VPP and IPP exerted discrepant inhibitory effects on this pathway. The EVs with RNase treatment lost the effects on VSMCs, indicating that the RNAs packed into vesicles may be a critical component. These results implied that VPP and IPP could alleviate Ang II-induced vascular dysfunction by modulating the EV-mediated transmission of RNAs between VECs and VSMCs.

Topics: Angiotensin II; Antihypertensive Agents; Cell Movement; Cell Proliferation; Coculture Techniques; Extracellular Vesicles; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-6; Myocytes, Smooth Muscle; Oligopeptides; RNA

Endothelial dysfunction is a common disorder of vascular homeostasis in hypertension characterized by oxidative stress, malignant migration, inflammatory response, and active adhesion response of endothelial cells. The extracellular vesicles (EVs), a vital participant in vascular cell communication, have been considered responsible for vascular disease progression. However, the potential mechanism of antihypertensive peptides against the EVs-induced endothelial dysfunction is still unclear. In this study, we investigated whether the antihypertensive peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) ameliorate the effects of EVs from Ang II-induced vascular smooth muscles (VSMCs) on the endothelial dysfunction. The dihydroethidium staining, wound healing assay, 3D cell culture, and co-culture with U937 monocyte were used to investigate the oxidant/antioxidant balance, migration, tube formation, and cell adhesion in EV-induced human umbilical vein endothelial cells. VPP and IPP treatment reduced the level of reactive oxygen species and EV-induced expression of adhesion molecules and restored the ability of tube formation by upregulating endothelial nitric oxide synthase expression. VPP and IPP reduced the protein levels of IL-6 to 227.34 ± 10.56 and 273.84 ± 22.28 pg/mL, of IL-1β protein to 131.56 ± 23.18 and 221.14 ± 13.8 pg/mL, and of MCP-1 to 301.48 ± 19.75 and 428.68 ± 9.59 pg/mL. These results suggested that the VPP and IPP are potential agents that can improve the endothelial dysfunction caused by EVs from Ang II-induced VSMCs.

Topics: Angiotensin II; Extracellular Vesicles; Human Umbilical Vein Endothelial Cells; Humans; Muscle, Smooth, Vascular; Oligopeptides; U937 Cells

There is great interest in developing naturally derived compounds, especially bioactive peptides with potential insulin sensitizing effects and/or preventing insulin resistance. Previously, we showed adipogenic and insulin mimetic actions of IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro), the milk-derived tripeptides on cultured preadipocytes, in addition to their previously characterized antihypertensive and anti-inflammatory functions. However, the effect of these peptides on insulin signaling is not known. Therefore, we examined IPP and VPP effects on insulin signaling in preadipocytes, a well-established model for studying insulin signaling. Our results suggested both peptides enhanced insulin signaling and contributed toward the prevention of insulin resistance in the presence of tumor necrosis factor (TNF). Inhibition of inflammatory mediator NF-kB under TNF stimulation was a likely contributor to the prevention of insulin resistance. VPP further enhanced the expression of glucose transporter 4 (GLUT4) in adipocytes and restored glucose uptake in TNF-treated adipocytes. Our data suggested the potential of these peptides in the management of conditions associated with impairments in insulin signaling.

Topics: Adipocytes; Adipogenesis; Animals; Cattle; Glucose; Glucose Transporter Type 4; Insulin; Insulin Resistance; Mice; Milk; NF-kappa B; NIH 3T3 Cells; Oligopeptides; Tumor Necrosis Factor-alpha

Bradykinin is the main player of the kallikrein-kinin system. Bradykinin-induced vasodilatation is age-dependent; this is believed to be associated with the level of expression of the two bradykinin receptors (BR1 and BR2) in the vasculature. The aim of this study was to clarify bradykinin-induced vascular reactivity of spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto rats (WKY) after 6 weeks' consumption of a drink containing bioactive tripeptides (Ile-Pro-Pro, Val-Pro-Pro and Leu-Pro-Pro). Two age groups were used: young (10 weeks at the end of experiment) and old (24 weeks). Blood pressure was monitored weekly by the tail-cuff method. After six weeks, vascular reactivity was assessed in vitro in mesenteric artery rings focusing on bradykinin induced activity. Blood pressure was lowered in old SHR after 6 weeks' tripeptide consumption compared to water drinking controls (P < 0.05). Blood pressure was lowered by peptide consumption also in old WKY (P < 0.05) but tripeptide consumption exerted no effect on the blood pressure of young animals. Old SHR suffered from endothelial and smooth muscle dysfunction which was not improved by these tripeptides. Interestingly, bradykinin caused vasoconstriction even in young SHR; this was blocked by a non-selective cyclooxygenase (COX) inhibitor but not by a B1 and B2 receptor antagonist. The expressions of mRNA of COX-1 and COX-2 in aorta were slightly upregulated in old SHR. ACE-1 activity in aorta and protein level in kidney, but not ACE-1 mRNA expression was upregulated in old animals (P < 0.05). To conclude, long-term feeding with a drink containing tripeptides lowers or prevents the age-associated increase in blood pressure in hypertensive and normotensive animals. ACE-1 activity, protein level but not mRNA expression are elevated in old animals. We also demonstrated that the vascular inflammation and dysfunction present in aged hypertensive animals cause bradykinin to induce vasoconstriction; this is not prevented by tripeptide feeding but involves the prostaglandin pathway.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Aorta; Blood Pressure; Bradykinin; Captopril; Cyclooxygenase 1; Hypertension; Kidney; Membrane Proteins; Mesenteric Arteries; Oligopeptides; Peptidyl-Dipeptidase A; Rats, Inbred SHR; Rats, Inbred WKY; Vasoconstriction; Vasodilator Agents

The bioactive peptides Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP) are believed to improve blood pressure and arterial function. To gain a better understanding of the mechanisms underlying the action of these peptides, we investigated their effects upon autonomic neurotransmission and blood pressure in spontaneously hypertensive rats (SHR). Both IPP and VPP caused a significant reduction in cutaneous arterial sympathetic nerve activity (CASNA) and reduced mean arterial pressure (MAP); however, both of these effects were eliminated following sub-diaphragmatic vagotomy. On the other hand, captopril, an angiotensin-converting enzyme inhibitor, reduced MAP without changing CASNA, and maintained this hypotensive effect following vagotomy. Moreover, the effects of IPP and VPP upon CASNA were observed following gastric administration but not by duodenal administration. These results suggest that IPP and VPP reduce CASNA via the stomach and afferent vagus nerve, thus causing reductions in MAP in SHR.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Autonomic Nervous System; Blood Pressure; Captopril; Chromosomal Proteins, Non-Histone; DNA Helicases; Drosophila Proteins; Hypertension; Oligopeptides; Rats, Inbred SHR; Synaptic Transmission; Transcription Factors; Vagotomy

The aim of this study was to identify and quantify the release of antihypertensive tripeptides valine-proline-proline (VPP) and isoleucine-proline-proline (IPP) during in vitro oro-gastro-intestinal (OGI) digestion of bovine skimmed milk. The experimental approach combined the recently developed harmonized static in vitro digestion (IVD) model and targeted mass spectrometry to monitor peptide generation. We first demonstrated that VPP and IPP are released from bovine milk proteins during in vitro OGI digestion at final concentrations of 354.3 ± 29.8 and 973.8 ± 155.7 μg/L, respectively. In silico analysis of cleavage sites and mass spectrometry revealed that tetrapeptides VPPF, IPPL, and IPPK are precursors of VPP and IPP. The release of other ACE-inhibitory peptides, such as FVAP, VAP, AW, and VY, was demonstrated, and their fate and the time course were investigated. This research underlines the suitability of an IVD system to study the release of short bioactive peptides during OGI transit.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Caseins; Cattle; Digestion; Gastrointestinal Tract; Humans; Kinetics; Models, Biological; Oligopeptides

Somatic angiotensin-converting enzyme (ACE) contains two active sites, the C- and N-domain, from which the C-domain is supposed to play a major role in blood pressure regulation and is therefore a promising pharmacological target to reduce blood pressure without side-effects. We report for the first time that tryptophan-containing dipeptides such as Ile-Trp or Val-Trp, which were recently found in food protein hydrolysates, are selective and competitive inhibitors for the C-domain with a selectivity factor of 40 and 70, respectively. Structure-activity studies showed that an N-terminal aliphatic amino acid and a tryptophan moiety in the P2' position are favourable structures for C-domain inhibition in dipeptides. In contrast, the lactotripeptides Ile-Pro-Pro and Val-Pro-Pro, which were widely used as ingredients for hypotensive food, showed a slight selectivity for the N-domain. Hence, tryptophan containing dipeptides are interesting ingredients for functional foods as a natural prevention for hypertension with reduced side effects due to its selective inhibition of the C-domain.

Topics: Angiotensin-Converting Enzyme Inhibitors; Blood Pressure; Body Mass Index; Catalytic Domain; Dipeptides; Humans; Hypertension; Oligopeptides; Peptidyl-Dipeptidase A; Protein Hydrolysates; Structure-Activity Relationship; Tryptophan

Milk derived tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) have shown promise as anti-hypertensive agents due to their inhibitory effects on angiotensin converting enzyme (ACE). Due to the key inter-related roles of hypertension, chronic inflammation and insulin resistance in the pathogenesis of metabolic syndrome, there is growing interest in investigating established anti-hypertensive agents for their effects on insulin sensitivity and inflammation. In this study, we examined the effects of IPP and VPP on 3T3-F442A murine pre-adipocytes, a widely used model for studying metabolic diseases. We found that both IPP and VPP induced beneficial adipogenic differentiation as manifested by intracellular lipid accumulation, upregulation of peroxisome proliferator-activated receptor gamma (PPARγ) and secretion of the protective lipid hormone adiponectin by these cells. The observed effects were similar to those induced by insulin, suggesting potential benefits in the presence of insulin resistance. IPP and VPP also inhibited cytokine induced pro-inflammatory changes such as reduction in adipokine levels and activation of the nuclear factor kappa B (NF-κB) pathway. Taken together, our findings suggest that IPP and VPP exert insulin-mimetic adipogenic effects and prevent inflammatory changes in adipocytes, which may offer protection against metabolic disease.

Topics: 3T3 Cells; Adipocytes; Adipogenesis; Adiponectin; Animals; Biomarkers; Cell Differentiation; Gene Expression Regulation; Inflammation; Lipid Metabolism; Mice; Milk; NF-kappa B; Oligopeptides; Signal Transduction; Transcription, Genetic; Tumor Necrosis Factor-alpha

Some food-derived peptides possess bioactive properties, and may affect health positively. For example, the C-terminal lacto-tri-peptides Ile-Pro-Pro (IPP), Leu-Pro-Pro (LPP) and Val-Pro-Pro (VPP) (together named here XPP) are described to lower blood pressure. The bioactivity depends on their availability at the site of action. Quantitative trans-organ availability/kinetic measurements will provide more insight in C-terminal tri-peptides behavior in the body. We hypothesize that the composition of the meal will modify their systemic availability. We studied trans-organ XPP fluxes in catheterized pigs (25 kg; n=10) to determine systemic and portal availability, as well as renal and hepatic uptake of a water-based single dose of synthetic XPP and a XPP containing protein matrix (casein hydrolyte, CasH). In a second experiment (n=10), we compared the CasH-containing protein matrix with a CasH-containing meal matrix and the modifying effects of macronutrients in a meal on the availability (high carbohydrates, low quality protein, high fat, and fiber). Portal availability of synthetic XPP was 0.08 ± 0.01% of intake and increased when a protein matrix was present (respectively 3.1, 1.8 and 83 times for IPP, LPP and VPP). Difference between individual XPP was probably due to release from longer peptides. CasH prolonged portal bioavailability with 18 min (absorption half-life, synthetic XPP: 15 ± 2 min, CasH: 33 ± 3 min, p<0.0001) and increased systemic elimination with 20 min (synthetic XPP: 12 ± 2 min; CasH: 32 ± 3 min, p<0.0001). Subsequent renal and hepatic uptake is about 75% of the portal release. A meal containing CasH, increased portal 1.8 and systemic bioavailability 1.2 times. Low protein quality and fiber increased XPP systemic bioavailability further (respectively 1.5 and 1.4 times). We conclude that the amount and quality of the protein, and the presence of fiber in a meal, are the main factors that increase the systemic bioavailability of food-derived XPP.

Topics: Animals; Dietary Supplements; Female; Kidney; Liver; Oligopeptides; Swine; Tissue Distribution

This study aimed at evaluating non-starter lactobacilli (NSLAB) isolated from cheeses for their proteolytic activity and capability to produce fermented milk enriched in angiotensin-converting enzyme (ACE)-inhibitory and antioxidant peptides. Preliminarily, 34 NSLAB from Parmigiano Reggiano (PR) and 5 from Pecorino Siciliano cheeses were screened based on their capacity to hydrolyze milk proteins. Two NSLAB strains from PR, Lactobacillus casei PRA205 and Lactobacillus rhamnosus PRA331, showed the most proteolytic phenotype and were positively selected to inoculate sterile cow milk. The fermentation process was monitored by measuring viable cell population, kinetic of acidification, consumption of lactose, and synthesis of lactic acid. Milk fermented with Lb. casei PRA205 exhibited higher radical scavenging (1184.83 ± 40.28 mmol/L trolox equivalents) and stronger ACE-inhibitory (IC50 = 54.57 μg/mL) activities than milk fermented with Lb. rhamnosus PRA331 (939.22 ± 82.68 mmol/L trolox equivalents; IC50 = 212.38 μg/mL). Similarly, Lb. casei PRA205 showed the highest production of ACE-inhibitory peptides Val-Pro-Pro and Ile-Pro-Pro, which reached concentrations of 32.88 and 7.52 mg/L after 87 and 96 h of milk fermentation, respectively. This evidence supports Lb. casei PRA205, previously demonstrated to possess characteristics compatible with probiotic properties, as a promising functional culture able to promote health benefits in dairy foods.

Topics: Animals; Antioxidants; Cattle; Cheese; Female; Fermentation; Lactic Acid; Lacticaseibacillus casei; Lactose; Milk; Milk Proteins; Oligopeptides; Peptides; Peptidyl-Dipeptidase A; Probiotics

Both endothelial dysfunction and arterial stiffness are surrogate markers of atherosclerosis and thus cardiovascular (CV) events. The milk-derived peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) inhibit angiotensin-converting enzyme, dilate blood vessels ex vivo and stimulate nitric oxide (NO) production in cells. In this study, we investigated the effects of either VPP or IPP on arterial function and on target organ damage in vivo. Male Wistar rats were treated with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 g l(-1)), L-NAME+VPP (0.3 g l(-1)) or L-NAME+IPP (0.3 g l(-1)) in their drinking water for 8 weeks. Plasma nitrite and nitrate (NOx) levels were significantly increased in normal Wistar rats after supplementation with either VPP or IPP but not in rats that were chronically treated with L-NAME. Acetylcholine-induced vasorelaxation in the thoracic aorta ring was impaired by L-NAME, whereas vasorelaxation was significantly greater in mice treated with L-NAME+VPP for 1 or 4 weeks or L-NAME+IPP for 4 weeks than in mice treated with L-NAME alone. Four weeks of treatment with either VPP or IPP attenuated the increase in pulse wave velocity (PWV) that was induced by L-NAME. Cardiac and renal damage were observed after 8 weeks of treatment with L-NAME, and either VPP or IPP attenuated this damage. These results show that VPP or IPP attenuates arterial dysfunction and suggest that milk-derived peptides might prevent CV damage.

Topics: Acetylcholine; Animals; Arteries; Blood Pressure; Enzyme Inhibitors; Male; Milk; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oligopeptides; Pulse Wave Analysis; Rats; Rats, Wistar; Vascular Diseases; Vasodilator Agents

Hypertension is a major global health issue which elevates the risk of a large world population to chronic life-threatening diseases. The inhibition of angiotensin-converting enzyme (ACE) is an effective target to manage essential hypertension. In this study, the fermentation properties (titratable acidity, free amino nitrogen, and fermentation time) and ACE-inhibitory (ACEI) activity of fermented milks produced by 259 Lactobacillus helveticus strains previously isolated from traditional Chinese and Mongolian fermented foods were determined. Among them, 37 strains had an ACEI activity of over 50%. The concentrations of the antihypertensive peptides, Ile-Pro-Pro and Val-Pro-Pro, were further determined by ultra performance liquid chromatography with quadrupole-time-of-flight mass spectrometry. The change of ACEI activity of the fermented milks of 3 strains exhibiting the highest ACEI activity upon gastrointestinal protease treatment was assayed. Fermented milks produced by strain H9 (IMAU60208) had the highest in vitro ACEI activity (86.4 ± 1.5%), relatively short fermentation time (7.5 h), and detectable Val-Pro-Pro (2.409 ± 0.229 µM) and Ile-Pro-Pro (1.612 ± 0.114 µM) concentrations. Compared with the control, a single oral dose of H9-fermented milk significantly attenuated the systolic, diastolic, and mean blood pressure of spontaneously hypertensive rats (SHR) by 15 to 18 mmHg during the 6 to 12 h after treatment. The long-term daily H9-fermented milk intake over 7 wk exerted significant antihypertensive effect to SHR, but not normotensive rats, and the systolic and diastolic blood pressure were significantly lower, by 12 and 10 mmHg, respectively, compared with the control receiving saline. The feeding of H9-fermented milk to SHR resulted in a significantly higher weight gain at wk 7 compared with groups receiving saline, commercial yogurt, and captopril. Our study identified a novel probiotic L. helveticus strain originated from kurut sampled from Tibet (China), which is a valuable resource for future development of functional foods for hypertension management.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Blood Pressure; China; Dairy Products; Diet; Fermentation; Hypertension; Lactobacillus helveticus; Male; Milk; Oligopeptides; Probiotics; Rats; Rats, Inbred SHR; Tibet; Yogurt

Lactobacillus helveticus H9 was isolated from traditionally fermented yak milk in Tibet (China) with the ability to produce the antihypertensive peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) during milk fermentation. To understand the changes in the protein expression of L. helveticus H9, proteome analysis was performed at 3 different growth stages, lag phase (pH 6.1), log phase (pH 5.1), and stationary phase (pH 4.5) using 2-dimensional electrophoresis (2-DE). Further analysis showed that 257 differential protein spots were found and 214 protein spots were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The cellular functions of the differentially expressed proteins were complex. Interestingly, the proteolytic system-related proteins aminopeptidase N (PepN), aminopeptidase E (PepE), endopeptidase O2 (PepO2), and oligopeptide transport system permease protein (OppC) were observed only on the maps of pH 5.1 and pH 4.5, which was consistent with the presence of angiotensin I-converting enzyme (ACE)-inhibitory peptides VPP and IPP during these 2 growth stages (log phase and stationary phase). These results, combined with a previous study of gene expression of the proteolytic system, led us to conclude that the Opp transport system, pepE, and pepO2 are likely related to the production of ACE-inhibitory peptides.

Topics: Animals; Bacterial Proteins; Cattle; Electrophoresis, Gel, Two-Dimensional; Endopeptidases; Female; Fermentation; Hypertension; Lactobacillus helveticus; Membrane Transport Proteins; Milk; Oligopeptides; Peptides; Peptidyl-Dipeptidase A; Proteolysis; Proteome; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tibet

Milk-derived peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), have angiotensin I-converting enzyme inhibitory activities and blood pressure-lowering effects. We examined the effects of these peptides on the development of atherosclerosis in apolipoprotein E-deficient [apoE(-/-)] mice. For 31 weeks, six-week-old male apoE(-/-) mice received a diet that included one of the following: fermented milk containing both VPP and IPP; casein hydrolysate containing both of these peptides; synthesized VPP; synthesized IPP; enalapril; captopril; or control diet. At the end of feeding, blood biochemistry, aortic atherogenesis, and gene expression by DNA microarray analysis were evaluated. There were no significant changes in the plasma lipid levels and 8-isoprostane, a marker of oxidative stress. The area ratio of intima to media in the aortic arch was significantly lower in the fermented milk, casein hydrolysate, synthesized VPP, enalapril, and captopril groups than in the control group. As is common with diets containing VPP and/or IPP, we observed reductions in mRNA expression of inflammatory cytokines, such as interleukin (IL)-6 and IL-1β, oxidized low-density lipoprotein receptor, and transcription regulators. These results suggest that a continuous intake of VPP and IPP might be beneficial for preventing atherosclerosis caused by hypercholesterolemia.

Topics: Animals; Aorta; Apolipoproteins E; Atherosclerosis; Disease Models, Animal; Gene Expression; Humans; In Vitro Techniques; Interleukin-1beta; Interleukin-6; Lipoproteins, LDL; Male; Mice; Mice, Knockout; Milk; Oligopeptides; Protein Hydrolysates

To understand high amount of production and detailed processing of antihypertensive peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), in Lactobacillus helveticus CM4 fermented milk, whole genome sequence of the CM4 strain was completed and compared to previously reported whole genome sequence of L. helveticus DPC4571. It revealed 2,028,493 bp of DNA sequence and encoding of 2174 open reading frames in the whole genome sequence with the highest homology to the genome sequence of L. helveticus DPC 4571. Comparative analysis focused on proteolytic enzymes between CM4 and DPC4571 strains revealed existence of 23 kinds of identical intracellular peptidase genes in both strains but no prtY type proteinase gene in DPC4571. Immunoblotting analysis with an antibody raised against the PrtY proteinase showed existence of the 45 kDa PrtY protein in CM4 but not in DPC4571 in the cell extracts. The cell wall-associated proteinase activity was higher in the CM4 than that in the DPC4571 throughout all fermentation period, and the amounts of VPP and IPP in CM4 and DPC4571 fermented milk were correlated with the proteinase activity on the cell wall. Moreover, slight difference of the β-casein hydrolysates by cell wall-associated extracellular proteinases between CM4 and DPC4571 cells was detected by a MALDI-TOF/TOF analysis. These results suggest that the extracellular proteinase activity might affect on the productivity of VPP and IPP in L. helveticus fermented milk and some peptidases might play important role in following precise processing to release VPP and IPP.

Topics: Amino Acid Sequence; Animals; Antihypertensive Agents; Cell Wall; DNA, Bacterial; Fermentation; Genome, Bacterial; Lactobacillus helveticus; Milk; Molecular Sequence Data; Oligopeptides; Open Reading Frames; Peptide Hydrolases; Peptides; Sequence Homology, Nucleic Acid

Lactobacillus helveticus can release the antihypertensive peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), from casein in fermented milk by a specific proteolytic system. To better understand the regulation of gene expression of the proteolytic enzymes thought to link to the processing of both antihypertensive peptides in L. helveticus, microarray analysis for whole gene expression in the presence and absence of added peptides in the fermented milk was studied. The productivity of both VPP and IPP in L. helveticus CM4 fermented milk was repressed by adding 2% quantity of Peptone as peptide mixture to the milk. Among the selected 13 amino acids, Gly, Ile, Leu, Phe, Met, Ser and Val were effective in the repression of the productivity of VPP and IPP in the fermented milk. The activity of the cell wall-associated proteinase, which may play a key role in the processing of the two antihypertensive peptides, was significantly repressed by the addition of the 2% quantity of Peptone into the fermented milk. By DNA microarray analysis it was found that prtH2 corresponding to the cell wall-associated proteinase gene, most of the endopeptidase genes such as pepE, pepO1, pepO2 and pepO3, most of the oligopeptide transporter genes, such as dppA2, dppB, dppC, dppD and dppF, most likely involved in the processing of VPP and IPP were down-regulated. These results suggest that amino acids released from milk peptides in the fermented milk might down-regulate the gene expressions of some of the proteolytic enzymes and may cause repression of the release of VPP and IPP in L. helveticus fermented milk.

Topics: Amino Acids; Animals; Antihypertensive Agents; Caseins; Endopeptidases; Fermentation; Gene Expression Regulation, Bacterial; Lactobacillus helveticus; Milk; Oligonucleotide Array Sequence Analysis; Oligopeptides; Peptides; Proteolysis; Transcriptome

The antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, were successfully detected in the aorta of spontaneously hypertensive rats after orally administering these peptides by a guanidine-thiocyanate treatment to prevent proteolysis. Cy3-labeled versions of both peptides were localized in the endothelial cells of arterial vessels in the rats. The accumulation of both peptides in the endothelial cells suggested in vivo inhibitory activity of the angiotensin I-converting enzyme.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Arteries; Blood Pressure; Carbocyanines; Endothelial Cells; Guanidines; Hypertension; Isotope Labeling; Male; Microscopy, Fluorescence; Oligopeptides; Peptidyl-Dipeptidase A; Rats; Rats, Inbred SHR; Thiocyanates

Milk casein-derived tripeptides, valyl prolyl proline (VPP), and isoleucyl prolyl proline (IPP) inhibit angiotensin-converting enzyme (ACE) and both fermented milk and proteolytic hydrolysates of milk casein containing these peptides exert blood pressure-lowering effects in animals and humans. On the top of these results, we have recently reported that the hydrolysate of milk casein containing both VPP and IPP improved the vascular endothelial function of subjects with stage I hypertension, enforcing us to elucidate the mechanism of the improvement of endothelial dysfunction by these peptides. For this purpose, we examined the effect of VPP and IPP on induction of nitric oxide (NO) production using cultured vascular endothelial cells and isolated arterial vessels. When both VPP and IPP were added to the medium of cultured endothelial cells at final concentrations of more than 100 nmol/l, the NO(x) (NO(2) and NO(3)) concentration in the medium was significantly higher than that of the control. Moreover, both VPP and IPP induced endothelium-dependent relaxation of isolated aortic rings, and these effects were inhibited by NO synthase inhibitors, K channel inhibitors, and bradykinin B2 receptor antagonists. These lines of results suggested that both VPP and IPP induced production of vasodilative substances including NO.

Topics: Animals; Aorta, Thoracic; Bradykinin B2 Receptor Antagonists; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Human Umbilical Vein Endothelial Cells; Humans; Male; Nitric Oxide; Nitric Oxide Synthase; Oligopeptides; Peptidyl-Dipeptidase A; Potassium Channel Blockers; Rats; Rats, Wistar; Receptor, Bradykinin B2; Time Factors; Vasodilation; Vasodilator Agents

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.

Topics: Aged; Antihypertensive Agents; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Circadian Rhythm; Female; Follow-Up Studies; Heart Rate; Humans; Male; Middle Aged; Oligopeptides

Milk casein-derived angiotensin-converting enzyme (ACE)-inhibitory tripeptides isoleucine-proline-proline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro) have been shown to have antihypertensive effects in human subjects and to attenuate the development of hypertension in experimental models. The aim of the present study was to investigate the effect of a fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro and plant sterols on already established hypertension, endothelial dysfunction and aortic gene expression. Male spontaneously hypertensive rats (SHR) with baseline systolic blood pressure (SBP) of 195 mmHg were given either active milk (tripeptides and plant sterols), milk or water ad libitum for 6 weeks. SBP was measured weekly by the tail-cuff method. The endothelial function of mesenteric arteries was investigated at the end of the study. Aortas were collected for DNA microarray study (Affymetrix Rat Gene 1.0 ST Array). The main finding was that active milk decreased SBP by 16 mmHg compared with water (178 (SEM 3) v. 195 (SEM 3) mmHg; P < 0.001). Milk also had an antihypertensive effect. Active milk improved mesenteric artery endothelial dysfunction by NO-dependent and endothelium-derived hyperpolarising factor-dependent mechanisms. Treatment with active milk caused mild changes in aortic gene expression; twenty-seven genes were up-regulated and eighty-two down-regulated. Using the criteria for fold change (fc) < 0.833 or > 1.2 and P < 0.05, the most affected (down-regulated) signalling pathways were hedgehog, chemokine and leucocyte transendothelial migration pathways. ACE expression was also slightly decreased (fc 0.86; P = 0.047). In conclusion, long-term treatment with fermented milk enriched with tripeptides and plant sterols decreases SBP, improves endothelial dysfunction and affects signalling pathways related to inflammatory responses in SHR.

Topics: Animals; Antihypertensive Agents; Blood Pressure; Caseins; Cultured Milk Products; Endothelium, Vascular; Gene Expression; Hypertension; Male; Nitric Oxide; Oligonucleotide Array Sequence Analysis; Oligopeptides; Peptidyl-Dipeptidase A; Phytosterols; Phytotherapy; Plant Extracts; Rats; Rats, Inbred Strains; Signal Transduction

Milk-based drinks containing casein-derived tripeptides isoleucine-proline-proline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro) have been shown to possess antihypertensive and vascular endothelium-protecting properties in hypertensive animal models. Furthermore in clinical intervention trials they reduce blood pressure and arterial stiffness. The exact mechanisms are not known, but inhibition of angiotensin converting enzyme 1 (ACE1) has been suggested mainly to mediate these beneficial effects. The present study investigated the in vitro effects of three tripeptides: Ile-Pro-Pro, Val-Pro-Pro and leqcine-proline-proline (Leu-Pro-Pro) on four renin-angiotensin system enzymes: ACE1, ACE2, chymase, and cathepsin G. Also their effects on arginase I, a critical enzyme in L-arginine-nitric oxide pathway, were studied. It was shown, apparently for the first time, that the inhibitory effects of Ile-Pro-Pro, Val-Pro-Pro and Leu-Pro-Pro on ACE1 at micromolar concentrations are competitive in nature. Therefore the efficacy of inhibition is largely dependent on the amount of substrate present. Inhibition of ACE2 and arginase I was reached only at concentrations three orders of magnitude greater. No inhibition of chymase and cathepsin G was observed by the tripeptides. The findings support the hypothesis that Ile-Pro-Pro, Val-Pro-Pro and Leu-Pro-Pro act favourably on blood pressure mainly by selective inhibition of ACE1.

Topics: Angiotensin-Converting Enzyme Inhibitors; Antihypertensive Agents; Arginase; Caseins; Cathepsin G; Chymases; Enzymes; Humans; Oligopeptides; Peptidyl-Dipeptidase A

Spectroscopic and structural elucidation of the peptides L-Valyl-L-Prolyl-L-Proline (1) and L-Isoleucyl-L-Prolyl-L-Proline (2) are reported on the basis of experimental linear-polarized IR-spectroscopy in solid-state, 1H-NMR data and DFT. Curiously, the experimental data shown that both peptides stabilized in solution and in solid-state neutral H2N-R-COOH form. Conformational analysis made, shown two strong intramolecular NH2-O=C-N(Amide) and O=C-OH-NH2 hydrogen bonds with lengths of 2.979 A and 2.475 A in (1) and 2.599 A and 2.507 A in (2) respectively. The presence of the Pro-Pro fold resulted to strong steric effect leading to the stabilization of free COOH and NH2 groups. The Erel values of zwitterion form are significant higher than the neutral forms with a difference of 1.2 and 0.9 kJ/mol. The manner of interaction of the peptides with angiotensin-I converting enzyme is proposed.

Topics: Angiotensin-Converting Enzyme Inhibitors; Antihypertensive Agents; Chromatography, High Pressure Liquid; Isoleucine; Models, Molecular; Normal Distribution; Nuclear Magnetic Resonance, Biomolecular; Oligopeptides; Proline; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Tandem Mass Spectrometry; Valine

Chick-pea (Cicer arietinum L.) cotyledons are unique source of aminopeptidase - 8-9 U/g cotyledons was observed using L-leucine-p-nitroanilide as substrate. The aminopeptidase was purified (65 kDa, pI 4.8 ) reaching a specific activity of 220 U/mg at pH 7.0-7.2 and 35-40 degrees C. The determined constant of specificity k(cat)/K(m) during hydrolysis of N-unsubstituted amino acid-p-nitroanilides showed a decrease order: Phe>Leu>Pro>Ile>Val>Ala. The enzyme was strongly inhibited by p-chloromercuribenzoic acid as well as in a competitive rate by the antihypertensive peptides Ile-Pro-Pro and Val-Pro-Pro.

Topics: Aminopeptidases; Cicer; Cotyledon; Hydrogen-Ion Concentration; Oligopeptides; Sequence Alignment; Substrate Specificity; Temperature

An active local renin-angiotensin system (RAS) has recently been found in the human eye. The aim of the present study was to compare the activities of central RAS enzymes (ACE1 and 2) in porcine ocular tissues, morphologically and physiologically close to the human eye. In addition, the effects of three ACE-inhibitory tripeptides on these enzymes were evaluated.. Enucleated fresh porcine eyes were used. Activities of ACE1 and ACE2 and their inhibition by bioactive tripeptides (Ile-Pro-Pro, Val-Pro-Pro, Leu-Pro-Pro) as well as by a standard ACE-inhibitor captopril were assayed in the vitreous body, the retina and the ciliary body using fluorometric detection methods.. Activity of ACE1 as well as ACE2 was found in all tissues evaluated. ACE1 activity was markedly higher in the ciliary body (3.7 +/- 0.7 mU/mg protein) than in retina (0.2 +/- 0.02 mU/mg), whereas ACE2 activities in the ciliary body (0.2 +/- 0.02 mU/mg) and retina (0.2 +/- 0.01 mU/mg) were at the same level. In the vitreous body ACE1 activity (8.2 +/- 0.31 nmol/min/mL) was manifold compared to that of ACE2 (0.1 +/- 0.02 nmol/min/mL). The tripeptides inhibited ACE1 at one-thousandth of the concentration needed to inhibit ACE2. All peptides studied evinced about equal inhibitory activities.. To our knowledge the present findings constitute the first evidence of ACE2 activity in the ciliary and vitreous bodies, in addition to previously described activity in the retina. The known favorable effects of ACE2 products vs. those of ACE1 suggest a counterbalancing interaction of these two enzyme homologues in physiological regulation of ocular circulation and pressure and possible protective role in certain ophthalmic disorders such as glaucoma and diabetic retinopathy.

Topics: Angiotensin-Converting Enzyme 2; Angiotensin-Converting Enzyme Inhibitors; Animals; Captopril; Ciliary Body; In Vitro Techniques; Oligopeptides; Peptidyl-Dipeptidase A; Retina; Swine; Vitreous Body

The angiotensin-converting enzyme (ACE) inhibitory activity and the concentration of the 2 ACE-inhibiting tripeptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) were studied during cheese ripening in 7 Swiss cheese varieties. The semi-hard cheeses Tilsiter, Appenzeller 1/4 fat, Tête de Moine, and Vacherin fribourgeois and the extra-hard and hard cheeses Berner Hobelkäse, Le Gruyère, and Emmentaler were investigated. Three loaves of each variety manufactured in different cheese factories were purchased at the beginning of commercial ripeness and investigated at constant intervals until the end of the usual sale period. Good agreement was found between ACE-inhibitory activity and the total concentration of VPP and IPP at advanced ripening stages. In most of the investigated varieties ACE-inhibitory activity and the concentration of the 2 tripeptides initially increased during the study period. A decline in the concentration of VPP and IPP was obtained toward the end of the investigated period for Tilsiter and Gruyère. The ratio of VPP/IPP decreased during ripening in all varieties with the exception of Emmentaler. However, large variations were observed among the cheese varieties as well as the individual loaves of the same variety. Chemical characterization of the investigated cheeses revealed that qualitative differences in the proteolysis pattern, not quantitative differences in the degree of proteolysis, are responsible for the observed variations in the concentrations of VPP and IPP. The presence of Lactobacillus helveticus in the starter culture was associated with elevated concentrations of VPP and IPP. The results of the present study show that concentrations of VPP and IPP above 100 mg/kg are attainable in semi-hard cheese varieties after ripening periods of about 4 to 7 mo and that stable concentrations of the 2 antihypertensive tripeptides can be expected over several weeks of cheese ripening.

Topics: Animals; Cheese; Food Handling; Oligopeptides; Peptidyl-Dipeptidase A; Time Factors

Topics: Angiotensin-Converting Enzyme Inhibitors; Oligopeptides; Peptidyl-Dipeptidase A; Substrate Specificity

The potential blood pressure lowering effect of fermented milk may involve inhibition of angiotensin-converting enzyme (ACE) by dairy lactotripeptides generated during milk fermentation, such as isoleucine-proline-proline (IPP) and valine-proline-proline (VPP). These peptides are weak ACE inhibitors in vitro but it remains unclear whether they inhibit ACE in vivo in humans.. To assess in vivo ACE inhibition in individuals given fermented milk over a 7-day period.. Twelve healthy normotensive men were given 330 ml of fermented milk once daily from day 1 to day 7 (IPP: 4.5 mg and VPP: 6.6 mg) and a single dose of 50 mg captopril on day 8. ACE inhibition was assessed in vivo by measuring plasma and urine AcSDKP and plasma active renin and in vitro by measuring plasma ACE activity using hippuryl-histidine-leucine. Plasma IPP/VPP concentrations were measured by LC/MS/MS.. Plasma IPP concentrations increased slightly and very transiently after fermented milk administration. Plasma VPP concentrations were below the limit of quantification. Fermented milk had no effect on plasma AcSDKP, ACE activity or active renin concentrations on days 1 or 7. Urine AcSDKP excretion underwent a small transient increase. In contrast, plasma and urine AcSDKP increased 7.7-fold and 70-fold, respectively, and plasma ACE activity decreased by 82.3 +/- 16.1% following captopril administration; plasma active renin concentration increased four-fold.. IPP and VPP were poorly absorbed and rapidly eliminated. They did not inhibit plasma or endothelial ACE in vivo at the selected doses and had no specific effect on the N-terminal or C-terminal ACE domains.

Topics: Animals; Catalytic Domain; Fermentation; Humans; Male; Milk; Oligopeptides; Peptidyl-Dipeptidase A; Reference Values; Renin

Many antihypertensive effects of angiotensin-I-converting enzyme (ACE) inhibitory peptides have been studied in spontaneously hypertensive rats (SHRs) and human, however, the mild actions of these peptides expressed by these consecutive uptakes are still not clear. Here, to understand the in vivo antihypertensive effects of well-characterized two peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), DNA microarray was used to analyze gene expression in SHRs fed these peptides for 5 days. By using an Affymetrix analyzer, gene profiling was performed in a target organ, the aorta, of SHRs after repeated administration of VPP and IPP for 5 days. The changes in gene expression were relatively mild; therefore, among the analyzed genes associated with blood pressure, those that showed changes over +/- 5% as compared to the control group were categorized as the renin angiotensin aldosterone system, vascular function, arachidonic acid system, blood coagulation system, and cytokines and growth factors. Significant and marked differences were detected for the endothelial nitric oxide synthase (eNOS) gene (1.89-fold, P<0.05) and the connexin 40 (gap junction 40) gene (2.81-fold, P<0.05). Administration of VPP and IPP led to a slight increase in the expression of the cyclooxigenase (COX-1) gene and a decrease in the expression of both the nuclear factor kappa B subunit (NF-kappaB) gene for vascular function and the peroxisome proliferator activator receptor gamma (PPARgamma) gene. Taken together, these results suggest that VPP and IPP function as ACE inhibitors in the aorta, where they may have a preventive role in cardiovascular function.

Topics: Administration, Oral; Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Aorta, Abdominal; Arachidonic Acid; Blood Coagulation; Blood Pressure; Blood Vessels; Cytokines; Endothelium; Gene Expression Regulation; Hypertension; Intercellular Signaling Peptides and Proteins; Male; Oligonucleotide Array Sequence Analysis; Oligopeptides; Peptidyl-Dipeptidase A; Polymerase Chain Reaction; Rats; Renin-Angiotensin System

The effect of chronic treatment with fermented milk products containing bioactive tripeptides and plant sterols on blood pressure and vascular function was investigated in spontaneously hypertensive rats (SHR). Six-weeks old male SHR (n=36) were randomized into 4 groups by body weight and blood pressure to receive either Lactobacillus helveticus fermented standard milk product (containing tripeptides Ile-Pro-Pro, Val-Pro-Pro and Leu-Pro-Pro), test product with enzymatically produced tripeptides without or with plant sterols or control product without the active constituents for 8 weeks. Systolic blood pressure (SBP) was measured weekly using the tail-cuff method. Thoracic aorta and mesenteric artery were excised for vascular response measurements. At the end, SBP values vs. control product group were: standard product group -14 mmHg (P<0.05), test product group -12 mmHg and test product +sterols group -7 mmHg. The average daily tripeptide dose was 2.8-5.2 mg/kg. Total serum cholesterol in the test product +sterols group tended to be lower than in the test product group (P=0.10) whereas serum plant sterol (campesterol, sitosterol) concentrations were higher (P<0.001). In conclusion, bioactive tripeptide-containing milk products attenuated the blood pressure development in SHR. The plant sterols did not improve this effect. Vascular responses did not markedly differ between the groups, except that endothelium-derived hyperpolarizing factor (EDHF) -related aortic relaxation was demonstrated in the test product +sterols group.

Topics: Acetylcholinesterase; Animals; Antihypertensive Agents; Arteries; Blood Pressure; Caseins; Cholesterol; Cultured Milk Products; Hypertension; Lactobacillus helveticus; Male; Oligopeptides; Phytosterols; Random Allocation; Rats; Rats, Inbred SHR; Time Factors

Tripeptides may possess bioactive properties. For instance, blood pressure lowering is attributed to the proline-rich tripeptides Ile-Pro-Pro (IPP), Leu-Pro-Pro (LPP), and Val-Pro-Pro (VPP). However, little is known about their absorption, distribution, and elimination characteristics. The aim of this study was to characterize the pharmacokinetic behavior of IPP, LPP, and VPP in a conscious pig model. Synthetic IPP, LPP, and VPP were administered intravenously or intragastrically (4.0 mg kg(-1) BW in saline) to 10 piglets (approximately 25 kg body weight) in the postabsorptive state. After intravenous dosing, the elimination half-life for IPP was significantly higher (P<0.001) than for LPP and VPP (2.5+/-0.1, 1.9+/-0.1, and 2.0+/-0.1 min, respectively). After intragastric dosing, however, the elimination half-lives were not significantly different between the peptides (9+/-1, 15+/-4, and 12+/-6 min, respectively). Maximum plasma concentrations were about 10 nmol l(-1) for the three tripeptides. The fraction dose absorbed was 0.077+/-0.010, 0.059+/-0.009, and 0.073+/-0.015%, for IPP, LPP, and VPP, respectively. Proline-rich tripeptides reach the blood circulation intact, with an absolute bioavailability of about 0.1% when administered via a saline solution. Because half-lives of absorption and elimination were maximally about 5 and 15 min, respectively, this suggests that under these conditions a bioactive effect of these tripeptides would be rather acute.

Topics: Animals; Antihypertensive Agents; Female; Oligopeptides; Swine

The contents of the 2 antihypertensive peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) were determined in 101 samples from 10 different Swiss cheese varieties using HPLC with subsequent triple mass spectrometry. In the category of extra hard and hard cheeses, the Protected Denomination of Origin cheeses Berner Alpkäse and Berner Hobelkäse, L'Etivaz à rebibes, Le Gruyère, Sbrinz, Emmentaler (organic and conventional) and in the category of semihard cheeses, the varieties Tilsiter, Appenzeller 1/4 fat and full fat, Tête de Moine, and Vacherin fribourgeois were screened in the study. The average concentration of the sum of VPP and IPP in the screened cheese varieties varied to a large extent, and substantial variations were obtained for individual samples within the cheese varieties. The lowest average concentration of the 2 tri-petides was found in L'Etivaz à rebibes (n = 3) at 19.1 mg/kg, whereas Appenzeller 1/4 fat (n = 4) contained the greatest concentration at 182.2 mg/kg. In individual samples, the total concentration of VPP and IPP varied between 1.6 and 424.5 mg/kg. With the exception of a 10-yr-old cheese, VPP was always present at greater concentrations than IPP. Milk pretreatment, cultures, scalding conditions, and ripening time were identified as the key factors influencing the concentration of these 2 naturally occurring bioactive peptides in cheese. The results of the present study show that various traditional cheese varieties contain, on average, similar concentrations of the 2 antihypertensive peptides to the recently developed fermented milk products with blood pressure-lowering property. This may serve as a basis for the development of a functional cheese with blood pressure-lowering property.

Topics: Angiotensin-Converting Enzyme Inhibitors; Antihypertensive Agents; Cheese; Oligopeptides; Switzerland; Tandem Mass Spectrometry

Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) are antihypertensive tripeptides isolated from milk fermented with Lactobacillus helveticus and inhibit angiotensin-converting enzyme (ACE). We investigated whether these peptides were generated from beta-casein by digestive enzymes and whether they were resistant to enzymatic hydrolysis, using an in vitro model. VPP and IPP were not generated from beta-casein by gastrointestinal enzymes; instead, a number of longer peptides including VPP and IPP sequences were detected. The fermentation step would therefore be necessary to produce these antihypertensive tripeptides. VPP and IPP themselves were hardly digested by digestive enzymes, suggesting that orally administered VPP and IPP remain intact in the intestine, retaining their activity until adsorption. The present study also demonstrated that various functional peptide sequences in beta-casein were resistant to gastrointestinal enzymes. There may be a strong correlation between the resistance of peptides to gastrointestinal digestion and their real physiological effects after oral administration.

Topics: Amino Acid Sequence; Animals; Antihypertensive Agents; Caco-2 Cells; Caseins; Digestion; Drug Stability; Fermentation; Gastrointestinal Tract; Humans; Milk; Oligopeptides; Peptide Fragments

Transepithelial transport of the ACE inhibitory peptides Ile-Pro-Pro and Val-Pro-Pro was studied in different models of absorption. Apparent permeability (P(app)) values for absorptive transport across Caco-2 monolayers were 1.0+/-0.9 x 10(-8) (Ile-Pro-Pro) and 0.5+/-0.1 x 10(-8)cms(-1) (Val-Pro-Pro). Ex vivo transport across jejunal segments in the Ussing chamber was 5-times (Ile-Pro-Pro) to 10-times (Val-Pro-Pro) higher with no significant differences (p>0.05) observed between both peptides. The peptidase inhibitor bestatin increased permeability for the absorptive direction for Ile-Pro-Pro by twofold. Neither a transepithelial pH gradient nor increased apical tripeptide concentration nor longitudinal localization of the intestinal segment influenced P(app) in the ex vivo experiments. Val-Pro-Pro transport across Peyer's patches, however, was 4-times higher (P(app)=21.0+/-9.3 x10(-8)cms(-1)) as compared to duodenum (P(app)=4.8+/-1.4 x 10(-8)cms(-1)). In the in situ perfusion experiments P(app) values varied greatly among different animals ranging from 0.5 to 24.0 x10(-8)cms(-1) (Ile-Pro-Pro) and from 1.0 to 15.6 x 10(-8)cms(-1) (Val-Pro-Pro). In summary, Caco-2 and ex vivo absorption models differ considerably regarding their peptide permeability. The in situ model seems to be less appropriate because of the observed large variability in peptide permeability. The results of this study demonstrate that the ACE inhibitory peptides Ile-Pro-Pro and Val-Pro-Pro are absorbed partially undegraded.

Topics: Absorption; Angiotensin-Converting Enzyme Inhibitors; Animals; Biological Transport; Caco-2 Cells; Cell Membrane Permeability; Cells, Cultured; Chromatography, Liquid; Diffusion Chambers, Culture; Humans; Intestinal Absorption; Jejunum; Models, Biological; Oligopeptides; Organ Culture Techniques; Rats

In recent years there has been an increasing body of literature describing the antihypertensive effects of peptides produced from milk protein. The tripeptides isoleucine-proline-proline (IPP) and valine-proline-proline (VPP), isolated from hydrolysed casein have been shown to lower blood pressure by inhibiting angiotensin I-converting enzyme (ACE). This has led to the use of these tripeptides, collectively referred to as lactotripeptide (LTP) as ingredients of functional foods intended to help control blood pressure. A programme of studies including a 90-day repeat-dose oral gavage toxicity study in the rat and an embryo-fetal (pre-natal) development study in the rabbit was conducted to ensure the safety of this ACE-inhibiting ingredient. In addition, a non-standard pre- and post-natal development study in the rat was performed. This study included direct dosing of the neonates, and was designed specifically to investigate renal development and to ensure that the bioactive peptides were not associated with the same type of fetopathy exhibited by ACE inhibiting drugs. These studies showed that there were no adverse effects of treatment at the highest doses tested.

Topics: Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Animals, Newborn; Blood Glucose; Chlorides; Cholesterol; Female; Fetal Development; Kidney; Male; Oligopeptides; Potassium; Rabbits; Random Allocation; Rats; Rats, Wistar; Reproduction; Sodium; Specific Pathogen-Free Organisms; Statistics, Nonparametric; Toxicity Tests, Chronic

The objective of these studies was to assess the toxicological potential of orally administered tripeptides in rats. The studies employed powdered L-valyl-L-prolyl-L-proline (VPP)- and L-isoleucyl-L-prolyl-L-proline (IPP)-containing test articles, including (1) powdered Lactobacillus helveticus-fermented milk (FM), (2) pasteurized casein hydrolysate (CH) generated by Aspergillus oryzae protease, and (3) synthesized VPP. All test articles were administered by oral gavage to male and female Sprague-Dawley rats. Specific goals of the single-dose and repeated-dose studies were to (1) identify doses that produce evidence of systemic and/or local (i.e., gastrointestinal) toxicity (e.g., lowest-observable-effect level [LOEL]); (2) estimate the maximally tolerated oral dose (MTD); and (3) identify specific target organs for toxicity of these tripeptides. Single doses of CH (2000 mg/kg), powdered FM (2000 or 4000 mg/kg), or VPP (40, 200, or 400 mg/kg) were administered 14 days prior to study termination. No treatment regimen caused either antemortem (gross observations, body weight, and food consumption parameters) or postmortem (necropsy) evidence of either systemic or local toxicity. In the repeated-dose study, powdered FM (0, 500, 1000, or 2000 mg/kg body weight [BW]/day) was administered by gastric gavage to male and female rats for 28 consecutive days. Antemortem evaluative parameters included gross observations, ophthalmic examinations, and clinical pathology (clinical chemistry, hematology, and urinalysis). Post mortem parameters included necropsy, determination of organ weights, and microscopic examination of major organs. There was neither in-life nor postmortem evidence that powdered FM administration caused physiological or toxicological changes. Under the conditions of these experiments, the single-dose LOEL of powdered FM, CH, and VPP were found to be greater than 4000, 2000, and 400 mg/kg, respectively. The results of the repeated-dose study do not support identification of a target organ for powdered FM toxicity. Similarly, there was no evidence to support establishment of either the LOEL or MTD; both being greater than 2000 mg/kg/day for up to 28 consecutive days.

Topics: Administration, Oral; Animals; Body Weight; Caseins; Cultured Milk Products; Dose-Response Relationship, Drug; Female; Hematocrit; Hemoglobins; Humans; Lactobacillus helveticus; Male; Oligopeptides; Organ Size; Powders; Rats; Rats, Sprague-Dawley; Sex Factors; Time Factors; Toxicity Tests; Urinalysis

The objective of these repeated-dose, 8-week studies was to assess the toxicological potential of a synthetic tripeptide, L-valyl-L-prolyl-L-proline (VPP), when administered to Charles River rats and Beagle dogs. Groups of 20 male and 20 female rats were fed powdered diets containing sufficient VPP to afford daily doses of 0, 2, 8, or 16 mg/kg body weight (BW)/day. Groups of five male and five female dogs were administered 0, 2, 8, or 16 mg/kg BW/day in hard gelatin capsules. Antemortem evaluative parameters for both species included grossly observable clinical signs, body weight and food consumption, clinical pathology (hematology, clinical chemistry, urinalysis), and ophthalmological examinations. Dogs also received electrocardiographic examinations. Postmortem evaluations in both species included complete necropsy, determination of major organ weights, and histopathological examination of specimens from approximately 50 organs and tissues. All rats and dogs survived to the scheduled termination of the studies and neither species exhibited evidence of VPP effects on appetite or body weight gain/maintenance. Ophthalmic examinations revealed occasional lens clouding in rats, but this occurred in all groups and was not attributable to VPP. Some clinical pathology parameters in both species were occasionally altered, but there was no evidence that this was dose-related. Electrocardiographic examinations in dogs revealed no VPP-associated changes. Mid- and high-dose male rats (but not females) had slightly reduced mean pituitary and kidney weight parameters, whereas mid- and high-dose females had slightly increased mean uterus:body weight ratios. There were no microscopic correlates for these minor changes. Ten percent to 20% of all female rats (but not males) exhibited corticomedullary mineralization of the kidney and gliosis of the optic nerve, and 10% to 20% of males (but not females) had thymic hemorrhage. Postmortem evaluations of dogs revealed no VPP-related effects on organ weights or either macro- or microscopic appearances of organs. The results of these studies provided no evidence of either local or systemic toxicity. Similarly, there was no evidence of neurotoxicity that might have been detected by the appearance of physical or behavioral changes during gross observations of animals. Although these results do not identify target organs for VPP toxicity, the no-observable-effect level and maximally tolerated dose are both greater than 16 mg/kg/day

Topics: Administration, Oral; Animals; Bilirubin; Blood Glucose; Body Weight; Calcium; Chemistry, Clinical; Corneal Opacity; Dogs; Dose-Response Relationship, Drug; Eating; Female; gamma-Glutamyltransferase; Hematology; Hemoglobins; Male; Monocytes; Oligopeptides; Organ Size; Ornithine Carbamoyltransferase; Rats; Sex Factors; Species Specificity; Specific Gravity; Time Factors; Toxicity Tests; Urinalysis

The objective of this multiple-dose toxicity study was to assess the toxicological potential of two tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP), when administered once daily for 91 consecutive days to rats. The test article, powdered casein hydrolysate (CH) known to contain 0.6% VPP plus IPP, was prepared using Aspergillus oryzae protease. Prior to administration to the rats by oral gavage, the test article was suspended in sterile water. Groups of 12 male and 12 female Charles River rats were administered once daily doses of 0, 40, 200, or 1000 mg of CH (0, 0.2, 1.2, or 6 mg VPP plus IPP/kg body weight [BW]). Antemortem evaluative parameters included gross observations of behavior and clinical signs; food consumption and body weight gains; ophthalmologic examinations; clinical pathology (hematology, clinical chemistry); and urinalysis. Postmortem parameters included determination of absolute and relative (to fasting body weight) organ weights and histopathological evaluation of approximately 50 organs and tissues from each animal. All rats survived until the scheduled termination of the study and no treatment-related clinical signs were observed. Food consumption was unaffected by administration of CH. All animals gained weight and there were no statistical differences between groups with respect to weight gains. There were no meaningful changes in hematological or coagulation parameters. Mid- and high-dose males (but not females) had slightly (< 2%) increased mean serum chloride concentrations, but because the difference was so small and it was observed in only one sex, the authors considered its association with CH administration to be doubtful. Urinalysis revealed the occasional presence of crystals, leukocytes, and epithelial cells in animals from all experimental groups. Similarly, ophthalmic changes (lenticular clouding) were observed in both control and dosed animals. Mean relative (to body weight) kidney weight was decreased by 8% in low-dose males and mean relative uterus weight was elevated 46% in low-dose females. Absolute organ weights were not affected. Only naturally occurring microscopic changes were observed in all groups and none could be attributed to CH administration. It was concluded that, under the conditions of these experiments, the maximally tolerated dose (MTD) and the no-observable-effect level (NOEL) for powdered CH administered once daily for 13 weeks was greater than 1000 mg/kg BW/d

Topics: Administration, Oral; Animals; Caseins; Cataract; Chlorides; Dose-Response Relationship, Drug; Eating; Female; Hematologic Tests; Kidney; Male; Oligopeptides; Organ Size; Powders; Rats; Rats, Sprague-Dawley; Sex Factors; Time Factors; Toxicity Tests; Urobilinogen; Uterus; Weight Gain

The objective of these studies was to assess the effects of the tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP), on reproductive capabilities of male and female rats. The specific goals of the experiments were (1) to determine the effects of orally administered tripeptides on (a) fertility and reproductive behavior in both sexes of rats, (b) embryo-fetal development in pregnant rats, and (c) pre- and postnatal development of rats exposed to tripeptides in utero and during lactation; and (2) to estimate the no-observable-adverse-effect doses of tripeptides in maternal and fetal rats. During the conduct of these classical segment I, II, and III studies, the test material was powdered Lactobacillus helveticus-fermented milk (FM), which contains the tripeptides, VPP and IPP. FM (0, 500, 1000 or 2000 mg/kg body weight [BW]/day--equivalent to 0, 0.8, 1.6, or 3.3 mg/kg BW/day of VPP plus IPP) was administered to males by oral gavage from 4 weeks prior to mating until sacrifice, and to females from 2 weeks prior to mating through day 20 of lactation. Evaluative parameters included monitoring grossly observable clinical signs; food consumption and body weight gains; mating behavior and fertility indices of both sexes; implantation and maintenance of embryos; sex ratio of live pups; fetal viability; incidences of external, visceral or skeletal variations; growth and behavioral development; as well as reproductive capabilities of F1 offspring exposed to FM during gestation and lactation. All animals were subjected to macroscopic examination at termination of their segment of the studies. Clinical signs, body weights, and food consumption were unaffected by administration of FM. During segment I, the test agent had no effect on estrus cycle, mating behavior, fertility index, or reproductive competence of either males or females. The results of segment II experiments revealed no effects of FM on postimplantation survival-loss, sex ratio or birth weights of live fetuses, and there was no evidence of treatment-associated developmental or teratological effects. During segment III, FM was without effect on pup viability, behavioral and sexual maturation, and reproductive capability of the F1 generation. Under the conditions of these experiments, the no-observable-adverse-effect level (NOAEL) of FM on reproductive performance in male and female rats is greater than 2000 mg/kg BW/day, the equivalent of 3.3 mg/kg BW/day of VPP plus IPP.

Topics: Animals; Animals, Newborn; Body Weight; Copulation; Cultured Milk Products; Dose-Response Relationship, Drug; Epididymis; Female; Fertility; Fetal Development; Kidney Pelvis; Lactobacillus helveticus; Male; Oligopeptides; Organ Size; Powders; Pregnancy; Rats; Rats, Sprague-Dawley; Reproduction; Sex Factors; Time Factors; Toxicity Tests

The objective of these in vivo experiments was to assess the mutagenic potential of tripeptides as reflected by the ability of the test compound to induce the formation of micronuclei in mouse polychromatic erythrocytes. The test agents used in these experiments were (1) powdered Aspergillus oryzae protease casein hydrolysate (CH) and (2) powdered Lactobacillus helveticus-fermented milk (FM). Both test agents contain two tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP). Male Sprague-Dawley rats (five per group) were administered doses of 0, 500, 1000, or 2000 mg (0, 3, 6, or 12 mg VPP plus IPP)/kg body weight (BW)/day CH by oral gavage for 2 days. Male CD-1 mice (six per group) received a single oral gavage dose of 0, 500, 1000, or 2000 mg (0, 0.8, 1.6 or 3.3 mg VPP plus IPP)/kg BW of FM. Positive-control agents were cyclophosphamide (10 mg/kg, intraperitoneal [i.p.]) in rats and mitocycin C (2 mg/kg, i.p.) in mice. Twenty-four hours after the second dose of CH, or the dose of cyclophosphamide to rats, or FM or mitocycin C to mice, bone marrow cells were fixed and examined for the presence of polychromatic erythrocytes (PCEs) and the presence or absence of micronucleated PCEs (MNPCEs). Administration of CH to rats and FM to mice produced neither changes in body weights nor signs of systemic toxicity. Similarly, neither CH nor FM caused statistically significant variations in the incidences of either PCEs or MNPCEs. Both positive-control agents caused unequivocal increases in the incidence of MNPCEs and cyclophosphamide significantly reduced the percent of rat erythrocytes appearing as PCEs. The results of these micronucleus assays conducted with either powdered CH or FM in rats and mice, respectively, show that neither form of the tripeptides possesses the potential to induce micronuclei formation in these rodent species.

Topics: Administration, Oral; Animals; Aspergillus oryzae; Bone Marrow Cells; Caseins; Cultured Milk Products; Cyclophosphamide; Dose-Response Relationship, Drug; Erythroblasts; Lactobacillus helveticus; Male; Mice; Mice, Inbred ICR; Micronucleus Tests; Mitomycin; Oligopeptides; Peptide Hydrolases; Powders; Rats; Rats, Sprague-Dawley; Toxicity Tests

The objective of this chromosomal aberration test was to assess the mutagenic potential of tripeptides by determining their ability to induce chromosomal aberrations in cultured Chinese hamster lung (CHL) cells. The test agents used in these experiments were (1) powdered casein hydrolysate (CH) and (2) powdered Lactobacillus helveticus-fermented milk (FM). Both test agents contain two tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP). CHL cells were cultured and exposed in the presence or absence of a rat hepatic metabolizing system (S9); CH or FM (1250, 2500, 5000 microg/ml of incubation medium); or positive-control agents, mitomycin C (0.1 or 0.05 microg/ml) or benzo(a)pyrene (20 microg/ml). In experiments with CH, cells were incubated for 6 h (either in the presence or absence of S9) or for 24 h (without S9). In experiments with FM, the cells were incubated for 6 h (either in the presence or absence of S9) or for 24 or 48 h (without S9). Neither short-term nor continuous exposure to either CH or FM caused the induction of significant changes in cell growth indices, incidences of chromosomal aberrations or the incidence of polyploids. Exposures of cells to mitomycin C and benzo(a)pyrene consistently resulted in the induction of the anticipated aberrant cells after either short-term or continuous exposures. The results of these assays support the conclusions that, under the conditions of these experiments, neither CH nor FM possesses demonstrable potential for the induction of cytotoxicity or clastogenesis.

Topics: Animals; Benzo(a)pyrene; Caseins; Cell Proliferation; Cell Survival; Cells, Cultured; Chromosome Aberrations; Cricetinae; Cricetulus; Cultured Milk Products; Dose-Response Relationship, Drug; Lactobacillus helveticus; Lung; Male; Microsomes, Liver; Mitomycin; Mutagenicity Tests; Oligopeptides; Powders; Rats; Rats, Sprague-Dawley

The objective of this study was to assess the mutagenic potential of a synthesized tripeptide, L-valyl-L-prolyl-L-proline (VPP), to induce mutational changes in Salmonella typhimurium LT2 strains TA1535, TA1537, TA98, and TA100, and Escherichia coli strain WP2uvrA in the classical Ames test protocol. Bacteria were exposed to plate concentrations of VPP of 0, 156.2, 312.5, 625, 1250, 2500, and 5,000 microg/plate in distilled water, in the presence and absence of Aroclor 1254-induced rat liver homogenate preparation (S9). Positive-control agents included sodium azide (TA100 and TA1535); 2-aminoanthracene (TA98, TA100, TA1535, TA1537, and WP2uvrA); 9-aminoacridine (TA1537); 2-nitrofluorene (TA98); and N-ethyl-N'-nitro-N-nitrosoguanidine (WP2uvrA) in DMSO. Incubations were conducted at 37 degrees C for about 48 h then revertant colonies were counted. All positive-control agents were consistently and unequivocally positive, but there was no evidence that VPP induced increases in the incidences of revertant colonies in any bacterial strain with and without metabolic activation. These findings were replicated in a second, confirmatory test performed with and without S9. The results of the experiments revealed no treatment-associated changes in the incidence of revertant colonies in any bacterial strain tested. These results support a conclusion that, under the experimental conditions described, there is no evidence that VPP possesses mutagenic potential.

Topics: Animals; Anthracenes; Dose-Response Relationship, Drug; Escherichia coli; Male; Microsomes, Liver; Mutagenicity Tests; Mutagens; Mutation; Oligopeptides; Rats; Rats, Sprague-Dawley; Salmonella typhimurium; Sodium Azide; Toxicity Tests

Milk fermented with Lactobacillus helveticus (L. helveticus) contains small peptides such as isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP), which inhibit the angiotensin converting enzyme (ACE). We investigated the effects of L. helveticus fermented milk whey (Lh-whey) and its components, sour milk whey, calcium and IPP and VPP peptides, on bone cells in vitro. An osteoblast assay was performed by determining the amount of deposited calcium as an index of bone formation in cultures of mouse osteoblasts formed from bone marrow-derived osteoblast precursor cells. An osteoclast assay was performed by determining the activity of tartrate-resistant acid phosphatase released into the culture medium in cultures of mouse osteoclasts formed from bone marrow-derived osteoclast precursor cells. The Lh-whey increased bone formation 1.3-1.4 times with the 1 x 10(-5), 1 x 10(-4) and 1 x 10(-3) solutions. The IPP and VPP peptides also demonstrated a significant 5-fold activation of bone formation in in vitro osteoblast cultures, whereas the sour milk whey and calcium had no effect. No significant effects were observed on osteoclasts in vitro with any of the study products. L. helveticus fermented milk whey contains bioactive components that increase osteoblastic bone formation in vitro. The effect may be due to the ACE-inhibitory IPP and VPP peptides, which showed a similar effect to that of the L. helveticus fermented milk whey.

Topics: Acid Phosphatase; Analysis of Variance; Angiotensin-Converting Enzyme Inhibitors; Animals; Bone Marrow Cells; Calcium Chloride; Cells, Cultured; Female; Fermentation; Isoenzymes; Lactobacillus; Mice; Milk; Milk Proteins; Oligopeptides; Osteoblasts; Osteoclasts; Osteogenesis; Tartrate-Resistant Acid Phosphatase; Whey Proteins

To purify and characterize a peptidase that can catalyse C-terminal processing of antihypertensive peptide from Lactobacillus helveticus CM4.. An endopeptidase which seems to process the carboxyl terminal end of two antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, was purified from Lactobacillus helveticus CM4 by four stages of column chromatography, using synthetic pro-peptide as a substrate. The molecular weight of the purified enzyme was estimated to be 67,000 by SEPHACRYL S-200 and 70,000 by SDS-PAGE analysis. The purified enzyme generated: (i) Val-Pro-Pro from Val-Pro-Pro-Phe-Leu and Val-Pro-Pro-Phe-Leu-Gln-Pro, and (ii) Ile-Pro-Pro from Ile-Pro-Pro-Leu-Thr and Ile-Pro-Pro-Leu-Thr-Gln-Thr, but theses peptides could not be generated from Val-Pro-Pro-Phe, Val-Pro-Pro-Phe-Leu-Gln, Ile-Pro-Pro-Leu and Ile-Pro-Pro-Leu-Thr-Gln. Part of the amino terminal sequence of the purified enzyme had homology to a previously reported pepO gene product.. These results suggest that the purified endopeptidase isolated in this study have an important role in the carboxyl terminal processing of two antihypertensive peptides in Lact. helveticus CM4.

Topics: Antihypertensive Agents; Endopeptidases; Lactobacillus; Oligopeptides; Substrate Specificity