The phospholipid fraction obtained from egg yolk reduces blood pressure increase induced by acute stress in spontaneously hypertensive rats *

Background. Despite continuous research, an improved understanding of the pathophysiology of hypertension and the development of new antihypertensive therapies, hypertension is still the most prevalent chronic cardiovascular disease (CVD) among adults in Western societies. Stress is a common factor related to cardiovascular morbidity. An increase in blood pressure is one of the most common reactions to stress. Chronic and acute stress have also been related to cardiovascular disorders. Polyunsaturated fatty acids (PUFAs) have attracted considerable interest as potential complementary therapy for the treatment of CVD, including hypertension. Egg yolk, rich in PUFAs and phospholipids, might be a good source of phospholipids and PUFAs.


Introduction
Eggs are an inexpensive and low-calorie food source which contains many substances indispensable for the development of a young organism, including minerals, proteins, n-3 polyunsaturated fatty acids (PUFAs), and phospholipids.However, eggs are also a source of cholesterol; therefore, the relationship between egg consumption and the risk of cardiovascular disease (CVD) remains controversial.Recently, the remarkable role of PUFAs in the prevention and treatment of CVD, including hypertension, has been described.The consumption of n-3 PUFAs reduces heart rate and systolic and diastolic blood pressure. 1,2The consumption of freshwater fish (300-600 g daily) is associated with increased plasma concentrations of n-3 PUFAs, lower blood pressure and lower plasma lipid concentrations. 3A metaanalysis of the effect of n-3 PUFAs in fish oil on blood pressure concluded that a higher consumption of n-3 PUFAs was associated with a higher reduction in blood pressure, particularly in hypertensive subjects and those with lipid disorders and atherosclerosis. 4Also, n-3 PUFA consumption improves endothelial function. 5The reduction in blood pressure may result from increased nitric oxide production, a reduced vasoconstrictive response to catecholamines and angiotensin I, improved vasodilatory responses, arterial compliance, 6,7 and reduced oxidative stress.
Hypertension is the most common chronic CVD in adults 8,9 and according to the WHO, it is the leading cause of death worldwide. 10,11Known hypertension risk factors include age, gender, obesity, lifestyle (diet and physical activity), 12,13 and additional cardiovascular risk factors (e.g., diabetes, dyslipidemia or insulin resistance). 14,15Chronic stress is linked to all cardiovascular disorders, including hypertension.Through the repeated occurrence of acute stressors and the activation of physiological stress-mediating systems, including the autonomic system, hypothalamicpituitary-adrenal axis and immune system, chronic stress causes an elevation in circulating catecholamine and cortisol levels.Circulating catecholamines acutely elevate blood pressure and contribute to vascular stiffening and cardiac hypertrophy. 16A stimulation of adrenergic activity increases sodium resorption, promoting blood pressure elevation. 17levated levels of cortisol additionally activate the reninangiotensin-aldosterone system, contribute to insulin resistance and hyperinsulinemia, and suppress nitric oxide, kallikrein and prostacyclin production.
Despite the progress in pharmacotherapy, hypertension treatment is still insufficient and the majority of patients worldwide have inadequately controlled blood pressure. 18lthough antihypertensive drugs play a fundamental role in blood pressure management, they may cause side effects; therefore, a number of studies have searched for food substances that can help reduce or prevent hypertension.In recent years, functional foods have attracted considerable interest as potential complementary therapies for the treatment of hypertension.The aim of this study was to evaluate the impact of a diet enriched with phospholipid fraction obtained from hen egg yolk on blood pressure in spontaneously hypertensive rats (SHRs) exposed to acute stress.
The study was conducted within the project "Innovative Production Technologies of Biopreparation Based on New Generation Eggs (OVOCURA)".We report here the primary findings of OVOCURA.

Material and methods
Male SHRs were purchased from Charles River Laboratories (Hamburg, Germany) and maintained under specific pathogen-free conditions in a temperature-controlled room (22 ±2°C) with a 12-hour light/dark cycle.All rats had ad libitum access to food and drinking water throughout the experiments.The experiments on animals were performed in accordance with relevant guidelines and regulations, and were conducted with the approval of the Local Ethical Commission at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław, Poland (approval No. 48/2012).Male, 7-week-old SHRs were divided into 2 groups: a study group on a supplemented diet (SHR/E group; n = 8) and a control group on a standard Labofeed B diet (SHR/C group; n = 8).The rats from the SHR/E group were given the supplemented diet for 12 weeks.
In our study, we used phospholipid fraction obtained from hen eggs and enriched naturally through feeding the hens substances such as PUFAs (DHA, for example); in this way, the yolk phospholipids were improved.The composition of the fraction was analyzed by gas chromatography/mass spectrometry (GC/MS).The egg phospholipid fraction is a mixture of phosphatidylcholine and phosphatidylethanolamine (the composition is detailed in Table 1).The diet of the study group after enrichment with phospholipid formulation contained 5% phosphatidylcholine, 0.65% omega-3 fatty acid and 0.95% omega-6 fatty acid, calculated globally.Spontaneously hypertensive rats (age of 15 week) were fitted with DSI telemetry devices (PA11P-C40, Data Sciences International, St. Paul, USA) at the 8 th week of diet supplementation.The animals were anesthetized with isoflurane (2-3%) through inhalation with a flow rate of 1.5 L/min.For all animals, the isoflurane concentration was adjusted by decreasing the dose to the minimum which would provide analgesia.An aseptic laparotomy was performed to expose the abdominal aorta.A catheter tip was inserted into the aorta and secured with medical glue.The body of the telemetry device was left inside the abdominal cavity and secured to the abdominal wall during suture closure of the incision.The system was programmed to collect data on systolic, mean and diastolic pressure.
Following the 12 th week of study, blood pressure was measured.The blood pressure measurements were taken before the stress procedure under normal conditions in each animal for 12 h.Subsequently, a model of acute stress with white noise was used.Blood pressure measurement was carried out during the exposure to stress.One 40-minute cycle of blood pressure monitoring in the presence of a stressor was carried out in each subject.
White noise was emitted at 100 dB through speakers located 30 cm above the cage.Blood pressure was measured simultaneously.The volume of sound was monitored by a decibel meter.After pressure measurement, the rats were anesthetized with isoflurane and sacrificed by cervical dislocation.Differences between the parameters under stress and before the procedure of acute stress (Δ) were calculated.Changes in the cardiovascular parameters (Δ) caused by acute stress were used as an indicator factor of stress level.

Statistical analysis
The data is expressed as mean ± standard error of the mean (SEM) for each parameter and group.The difference between means for independent variables was analyzed using the Student's t-test or Mann-Whitney U test, while differences within one group (dependent variables) were analyzed using the dependent Student's t-test or Wilcoxon test.A p-value <0.05 was considered statistically significant.The statistical analysis was performed with STATISTICA v. 10.0 software (StatSoft Inc., Tulsa, USA).

Results
Twelve-hour measurement intervals under normal conditions showed that the mean pressure, systolic pressure and diastolic pressure were significantly lower in the SHRs fed an enriched diet than in the control group (159/106 mm Hg vs 174/117 mm Hg).In all groups, the acute stress exposure evoked a significant increase in mean pressure, systolic pressure, diastolic pressure, and heart rate, whereas pulse pressure significantly increased only in the control group (Table 2).Moreover, a stress-induced increase in mean pressure (ΔP) and systolic pressure (ΔSP) was significantly lower in the study group than in the control group (ΔP: 23.42 mm Hg vs 36.86 mm Hg; ΔSP: 24.95 mm Hg vs 40.02 mm Hg).The increase in these parameters caused by stress was less pronounced in rats fed a diet enriched with PUFAs.However, the difference in stress-induced diastolic pressure changes (ΔDP) between groups did not reach statistical significance (p = 0.07) due to the small number of animals (Table 3).

Discussion
There has been an increased focus on identifying the natural components of foods which could be used in the prevention and treatment of hypertension.Grains, vegetables, fruits, dairy products, meat, chicken, eggs, fish, soybean, tea, wine, mushrooms, and lactic acid bacteria are various natural food sources with potential antihypertensive effects. 19Blood pressure is regulated through complex mechanisms, including the modification of angiotensinconverting enzyme (ACE) activity and changes in oxidative  status.Antioxidants may decrease arterial blood pressure by decreasing oxidative stress, thus preserving the activity of nitric oxide synthase (NOS) and increasing nitric oxide bioavailability.It was previously demonstrated that egg consumption may decrease oxidative stress levels; thus, our results are in agreement with those reported by Jahandideh et al. 20 Peptides obtained from egg white, such as novokinin, may act as a potent hypotensive factor through the AT(2) receptor. 21Fatty acids from fish oil are known to decrease blood pressure. 22It has been shown in clinical trials that n-3 PUFAs reduce the incidence of CVD and sudden cardiac death, and they decrease blood pressure. 23It has been demonstrated that dietary compounds rich in PUFAs reduce pressor responses of the mesenteric artery to norepinephrine. 24It is well-established that egg yolk contains peptides that demonstrate antioxidant, ACE-inhibitory and antidiabetic (α-glucosidase and DPP-IV inhibitory) activities. 25owever, there has not been any clinical experience with the use of phospholipid fraction obtained from hen egg yolk as a mean to control blood pressure.Hen eggs are a relatively inexpensive source of not only fatty acids but also of amino acids and arginine, a precursor to nitric oxide. 26In our study, rats fed a diet enriched with phospholipid fraction obtained from egg yolk had decreased blood pressure compared to the control group.The decrease in blood pressure was even more significant in rats exposed to acute stress.
Stress has become a prevalent part of people's lifestyle; therefore, the effect of stress on blood pressure is of increasing relevance and importance.We used noise as a stressor because it is a constant component of modern life and is considered to be a hypertensive factor.For what is likely the first time, advanced cardiovascular telemetry devices were utilized to evaluate the response to stress.The use of a modern experimental model which allows blood pressure monitoring in conscious rats via telemetry seems to be an important step in identifying the effects of phospholipid fraction on stress and blood pressure.It is well-known that acute stress causes a rise in blood pressure and heart rate.This new method of stress level evaluation might be used in the pre-clinical selection of bioactive products with the highest anti-stress potential.
Our results showing the influence of PUFAs on blood pressure are in agreement with the data of Jayasooriya et al., who showed that PUFA supplementation reduces hypertension in rats (animals with high ANG II activity). 27Thus, our findings in this study expand on existing evidence that PUFAs decrease blood pressure. 28Moreover, we observed that a diet enriched with PUFAs reduces the increase in blood pressure evoked by acute stress.We observed that phospholipid fraction obtained from hen egg yolk rich in PUFAs acts as a hypotensive agent and that it could be used in the prevention or treatment of hypertension evoked or exacerbated by acute stress.This finding suggests that egg phospholipid fraction could play an important role in the prevention of blood pressure rise related to stress.
Moreover, if we suppose that blood pressure change is an indicator of stress level, this study would suggest that phospholipids demonstrate anti-stress activity.The use of a hypertensive animal model probably enhances this effect.However, further studies with different stressors and stress models are needed in order to confirm these early results.In the last decade, there has been increasing interest in foods that promote health and could prevent hypertension and CVD.Chicken eggs have been demonstrated to decrease blood pressure; therefore, they can be considered hypotensive agents because they may become a part of a daily diet as functional food.

Conclusions
The results of this study indicate that the phospholipid fraction rich in fatty acids which is obtained from egg yolk reduces the negative response of the cardiovascular system to acute stress in SHRs.Since the study demonstrated the beneficial effects of phospholipid fraction on blood pressure in rats exposed to stress, the implications for patients need to be tested in further studies.

Table 2 .
The mean pressure, systolic pressure, diastolic pressure, pulse pressure, and heart rate under normal conditions and under acute stress in both groups: the control group (SHR/C) and the study group (SHR/E)