Perspective: Human Milk Composition and Related Data for National Health and Nutrition Monitoring and Related Research.

National health and nutrition monitoring is an important federal effort in the United States and Canada, and the basis for many of their nutrition and health policies. Understanding of child exposures through human milk (HM) remains out of reach due to lack of current and representative data on HM's composition and intake volume. This article provides an overview of the current national health and nutrition monitoring activities for HM-fed children, HM composition (HMC) and volume data used for exposure assessment, categories of potential measures in HM, and associated variability factors. In this Perspective, we advocate for a framework for collection and reporting of HMC data for national health and nutrition monitoring and programmatic needs, including a shared vision for a publicly available Human Milk Composition Data Repository (HMCD-R) to include essential metadata associated with HMC. HMCD-R can provide a central, integrated platform for researchers and public health officials for compiling, evaluating, and sharing HMC data. The compiled compositional and metadata in HMCD-R would provide pertinent measures of central tendency and variability and allow use of modeling techniques to approximate compositional profiles for subgroups, providing more accurate exposure assessments for purposes of monitoring and surveillance. HMC and related metadata could facilitate understanding the complexity and variability of HM composition, provide crucial data for assessment of infant and maternal nutritional needs, and inform public health policies, food and nutrition programs, and clinical practice guidelines.

Phosvitin Derived Phospho-Peptides Show Better Osteogenic Potential than Intact Phosvitin in MC3T3-E1 Osteoblastic Cells.

Phosphorylated proteins from food sources have been investigated as regulators of bone formation with potential benefits in treating osteoporosis. Egg, a cheap and nutritious food, is also the source of various proteins and bioactive peptides with applications in human health. Egg yolk is rich in phosvitin, the most phosphorylated protein in nature. Phosvitin has been shown to improve bone health in experimental animals, although the molecular mechanisms and its specific effects on bone-forming osteoblastic cells are incompletely understood. Previous work in our group has identified pancreatin-generated phosvitin phospho-peptides (PPP) as a potential source for bioactive peptides. Given this background, we examined the roles of both phosvitin and PPP in the function of osteoblastic cells. Our results demonstrated their potential to improve bone health by promoting osteoblast differentiation and proliferation, suppressing osteoclast recruitment and the deposition of extracellular matrix, although PPP appeared to demonstrate superior osteogenic functions compared to phosvitin alone.

Food-Derived Bioactive Peptides in Human Health: Challenges and Opportunities.

Recent scientific evidence suggests that food proteins not only serve as nutrients, but can also modulate the body's physiological functions. These physiological functions are primarily regulated by some peptides that are encrypted in the native protein sequences. These bioactive peptides can exert health beneficial properties and thus are considered as a lead compound for the development of nutraceuticals or functional foods. In the past few decades, a wide range of food-derived bioactive peptide sequences have been identified, with multiple health beneficial activities. However, the commercial application of these bioactive peptides has been delayed because of the absence of appropriate and scalable production methods, proper exploration of the mechanisms of action, high gastro-intestinal digestibility, variable absorption rate, and the lack of well-designed clinical trials to provide the substantial evidence for potential health claims. This review article discusses the current techniques, challenges of the current bioactive peptide production techniques, the oral use and gastrointestinal bioavailability of these food-derived bioactive peptides, and the overall regulatory environment.

Egg White-Derived Tripeptide IRW (Ile-Arg-Trp) Is an Activator of Angiotensin Converting Enzyme 2.

Angiotensin converting enzyme 2 (ACE2) plays beneficial roles in the renin angiotensin aldosterone system. Our previous studies indicated that egg white-derived antihypertensive peptide IRW (Ile-Arg-Trp) could upregulate ACE2 mRNA level in mesenteric artery of spontaneously hypertensive rats (SHRs), suggesting the potential of IRW as an in vivo ACE2 activator. In this study, the effects of IRW on activity and protein expression of ACE2 were investigated. Results indicated that IRW activated human recombinant ACE2 with an EC50 value of 7.24 × 10-5 M. In rat aortic vascular smooth muscle cell line A7r5 cells, IRW treatment (50 µM) significantly increased the expression and activity of ACE2. Oral administration of IRW to SHRs upregulated ACE2 protein levels in kidney and aorta. Molecular docking study suggested that IRW might activate ACE2 through interaction with the subdomain I near the active site through hydrogen bonds. Overall, this study established IRW as the first food-derived ACE2 activating peptide.

Milk-Derived Tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) Enhance Insulin Sensitivity and Prevent Insulin Resistance in 3T3-F442A Preadipocytes.

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.

Purification and characterization of antioxidant peptides from cooked eggs using a dynamic in vitro gastrointestinal model in vascular smooth muscle A7r5 cells.

Antioxidant peptides derived from food sources are considered as safer alternatives to commercially available antioxidant drugs. As one of the most abundant protein sources, hen's egg proteins were extensively used to produce antioxidant peptides by enzymatic hydrolysis. Our previous work indicated that gastrointestinal digestion of cooked eggs significantly increased the antioxidant activity due to hydrolysis of egg proteins. To characterize the responsible antioxidant peptides, cooked eggs were digested in a simulated in vitro model of human gastro-intestinal digestion. Prepared digests were fractionated with FPLC (Fast Protein Liquid Chromatography) and RP-HPLC (Reverse-Phase High-Performance Liquid Chromatography) and the antioxidant activity was determined in A7r5 cells (vascular smooth muscle cell line). Further identification of peptides from peptide fractions with the highest antioxidant activity was carried out using LC-MS/MS. Four peptides derived from ovalbumin, DSTRTQ (48-53), DKLPG (61-65), DVYSF (96-100), and ESKPV (205-209), were identified; of which DKLPG did not show antioxidant activity in cells. Enzyme cleave analysis suggested that these four peptides were likely released from ovalbumin only by pepsin non-specific cleaves. It is postulated that egg consumption may exert protection against oxidative stress on human health due to release of antioxidant peptides during digestion.

Egg white hydrolysate shows insulin mimetic and sensitizing effects in 3T3-F442A pre-adipocytes.

Insulin resistance and inflammation in adipose tissue is a key mechanism underlying metabolic syndrome, a growing health problem characterized by diabetes, obesity and hypertension. Previous work from our research group has demonstrated the potential of egg white ovotransferrin derived bioactive peptides against hypertension, oxidative stress and inflammation in vitro and in vivo. Egg white hydrolysate (EWH) has also shown anti-hypertensive effects in spontaneously hypertensive rats. Given the interplay among hypertension, inflammation, oxidative stress and metabolic syndrome, the objective of the study was to test the EWH on differentiation, insulin signaling and inflammatory responses in 3T3-F442A pre-adipocytes. Our study suggested that EWH could promote adipocyte differentiation as shown by increased lipid accumulation, increased release of adiponectin and upregulation of peroxisome proliferator associated receptor gamma (PPARγ) and CCAAT/ enhancer binding protein alpha (C/EBP-α). In addition to enhanced insulin effects on the upregulation of protein kinase B/Akt phosphorylation, EWH treatment increased extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation to a level similar to that of insulin, indicating insulin sensitizing and mimetic properties of the EWH. EWH further attenuated cytokine induced inflammatory marker; cyclooxygenase -2 (COX-2) by 48.78%, possibly through the AP-1 pathway by down regulating c-Jun phosphorylation in adipocytes. Given the critical role of adipose in the pathogenesis of insulin resistance and metabolic syndrome, EWH may have potential applications in the prevention and management of metabolic syndrome and its complications.

Modulatory Effects of Egg White Ovotransferrin-Derived Tripeptide IRW (Ile-Arg-Trp) on Vascular Smooth Muscle Cells against Angiotensin II Stimulation.

The renin angiotensin system (RAS) is a key mediator of blood pressure regulation. Angiotensin II (Ang II), the active component of RAS, is a potent vasoconstrictor that also causes abnormal proliferation, oxidative stress, and inflammation in vascular smooth muscle cells (VSMCs) that contribute to atherosclerotic changes. Egg white ovotransferrin-derived tripeptide IRW (Ile-Arg-Trp) was previously shown to exert antihypertensive effect by reducing Ang II synthesis as well as endothelial cell inflammation and endothelial dysfunction. However, the effects of IRW on VSMCs are still unclear. In the present study, we evaluated the antiproliferative, antioxidant, and anti-inflammatory effects of IRW on VSMCs in the presence of Ang II stimulation. It was found that IRW treatment could attenuate Ang II-stimulated proliferation, superoxide production, and inflammation in VSMCs. These beneficial effects appeared to involve modulation of the NF-κB pathway. These findings could further our understanding on the antihypertensive mechanism of IRW beyond vascular endothelium.

Low-molecular-weight fractions of Alcalase hydrolyzed egg ovomucin extract exert anti-inflammatory activity in human dermal fibroblasts through the inhibition of tumor necrosis factor-mediated nuclear factor κB pathway.

Ovomucin is a mucin-like protein from egg white with a variety of biological functions. We hypothesized that ovomucin-derived peptides might exert anti-inflammatory activity. The specific objectives were to test the anti-inflammatory activities of different ovomucin hydrolysates and its various fractions in human dermal fibroblasts, and to understand the possible molecular mechanisms. Three ovomucin hydrolysates were prepared and desalted; only the desalted Alcalase hydrolysate showed anti-inflammatory activity. Desalting of ovomucin hydrolysate enriched the proportion of low-molecular-weight (MW) peptides. Indeed, ultrafiltration of this hydrolysate displayed comparable anti-inflammatory activity in dermal fibroblasts, indicating the responsible role of low-MW bioactive peptides in exerting the beneficial biological function. The anti-inflammatory activity of low-MW peptides was regulated through the inhibition of tumor necrosis factor-mediated nuclear factor κ-light-chain-enhancer of activated B cells activity. Our study demonstrated that both peptide composition and MW distribution play important roles in anti-inflammatory activity. The low-MW fractions prepared from ovomucin Alcalase hydrolysate may have potential applications for maintenance of dermal health and treatment of skin diseases.

Antioxidant Peptides Identified from Ovotransferrin by the ORAC Method Did Not Show Anti-Inflammatory and Antioxidant Activities in Endothelial Cells.

Oxygen radical absorbance capacity (ORAC) is a widely used method of measuring antioxidant capacities of various antioxidant components. Surprisingly, 16 antioxidant peptides previously identified from egg protein ovotransferrin using the ORAC method did not show any anti-inflammatory and antioxidant activities in cells. After simulated gastro-intestinal digestion (GID), several peptide digests significantly reduced the expression of tumor necrosis factor-α (TNF-α)-induced pro-inflammatory intercellular cell adhesion molecule-1 (ICAM-1) by 65.7 ± 10.4% and vascular cell adhesion molecule-1 (VCAM-1) by 53.5 ± 9.6% to 61.0 ± 14.5%, but only GWNI reduced TNF-α-activated superoxide generation by 71.0 ± 12.9% when tested with dihydroethidium (DHE) assay. Mass spectrometer analysis identified two new peptides, GWN and GW, in the GWNI digest; however, only GW reduced TNF-α-induced VCAM-1 expression (64.3 ± 20.6%) significantly compared to the TNF-α treated cells. Our study suggested that ORAC lacked biological relevance in assessing bioactive peptides.

Analysis of G-Protein Coupled Receptor 30 (GPR30) on Endothelial Inflammation.

The female sex hormone estrogen (the most common form 17-ß-estradiol or E2) is known to have both anti-inflammatory and pro-inflammatory effects. Given the diversity of estrogen responses mediated through its three distinct receptors, namely, estrogen receptor α (ERα), ERß, and the G-protein coupled receptor 30 (GPR30), it is plausible that different receptors have specific modulatory effects on inflammation in different tissues. We have shown that activation of GPR30 exerted anti-inflammatory effects as demonstrated by significant attenuation of tumor necrosis factor (TNF)-mediated upregulation of adhesion molecules in isolated human umbilical vein endothelial cells. Interestingly, estrogen alone had no such effect and blockade of classical ERs restored the anti-inflammatory effect, suggesting that this effect was dependent on GPR30 and opposed to classical ERs. These findings were further validated by the negation of anti-inflammatory GPR30 effects by classical ER agonists. This chapter focuses on multiple pharmacological options to activate GPR30 and the use of TNF activated endothelial cells as a model system for inflammatory response as assessed by adhesion molecule detection through western blotting.

Milk-derived tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) promote adipocyte differentiation and inhibit inflammation in 3T3-F442A cells.

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.

Beneficial effects of simulated gastro-intestinal digests of fried egg and its fractions on blood pressure, plasma lipids and oxidative stress in spontaneously hypertensive rats.

BACKGROUND: We have previously characterized several antihypertensive peptides in simulated digests of cooked eggs and showed blood pressure lowering property of fried whole egg digest. However, the long-term effects of this hydrolysate and its fractions on blood pressure are not known. Therefore, the objectives of the study were to determine the effects of long term administration of fried whole egg hydrolysate and its fractions (i.e. egg white and egg yolk) on regulation of blood pressure and associated factors in cardiovascular disease such as plasma lipid profile and tissue oxidative stress. METHODS AND RESULTS: We used spontaneously hypertensive rats (SHR), an animal model of essential hypertension. Hydrolysates of fried egg and its fractions were prepared by simulated gastro-intestinal digestion with pepsin and pancreatin. 16-17 week old male SHRs were orally administered fried whole egg hydrolysate, non-hydrolyzed fried whole egg, egg white hydrolysate or egg yolk hydrolysates (either defatted, or not) daily for 18 days. Blood pressure (BP) and heart rate were monitored by telemetry. Animals were sacrificed at the end of the treatment for vascular function studies and evaluating plasma lipid profile and tissue oxidative stress. BP was reduced by feeding fried whole egg hydrolysate but not by the non-hydrolyzed product suggesting a critical role for in vitro digestion in releasing anti-hypertensive peptides. Egg white hydrolysate and defatted egg yolk hydrolysate (but not egg yolk hydrolysate) also had similar effects. Reduction in BP was accompanied by the restoration of nitric oxide (NO) dependent vasorelaxation and reduction of plasma angiotensin II. Fried whole egg hydrolysate also reduced plasma levels of triglyceride although it was increased by the non-hydrolyzed sample. Additionally the hydrolyzed preparations attenuated tissue oxidative stress. CONCLUSION: Our results demonstrate that fried egg hydrolysates exert anti-hypertensive effects, improve plasma lipid profile and attenuate tissue oxidative stress in vivo.

Coiled-coil domain containing 3 (CCDC3) represses tumor necrosis factor-α/nuclear factor κB-induced endothelial inflammation.

Coiled-coil domain containing 3 (CCDC3) is a newly identified secretory protein that is expressed in vascular endothelial cells (ECs) and adipose tissues. Here, we investigate the role of CCDC3 in tumor necrosis factor (TNF)-α-induced inflammatory response in ECs. Our results show that stable overexpression of CCDC3 decreases, while stable knockdown of the endogenous CCDC3 increases TNF-α-induced expression of vascular cell adhesion molecule-1 (VCAM-1) at the mRNA and protein level in ECs. The IκB kinase inhibitor Bay 11-7082 completely blocks TNF-α-induced expression of VCAM-1, confirming that TNF-α-induced expression of VCAM-1 in ECs is nuclear factor κB (NF-κB) dependent. Stable overexpression of CCDC3 decreases TNF-α-induced p65 and p50 nuclear translocation and NF-κB transcriptional activity, suggesting that CCDC3 inhibits TNF-α-induced NF-κB activation in ECs. Similar to CCDC3 overexpression, both CCDC3-containing conditioned medium (CM) and purified CCDC3 decrease TNF-α-induced expression of VCAM-1 in receiving ECs, suggesting a paracrine/autocrine function of CCDC3. Interestingly, CCDC3 in CM can enter the receiving ECs. Taken together, our work demonstrates that CCDC3 represses TNF-α/NF-κB-induced pro-inflammatory response in ECs, providing an insight into the functional role of CCDC3.

Food-derived bioactive peptides on inflammation and oxidative stress.

Chronic diseases such as atherosclerosis and cancer are now the leading causes of morbidity and mortality worldwide. Inflammatory processes and oxidative stress underlie the pathogenesis of these pathological conditions. Bioactive peptides derived from food proteins have been evaluated for various beneficial effects, including anti-inflammatory and antioxidant properties. In this review, we summarize the roles of various food-derived bioactive peptides in inflammation and oxidative stress and discuss the potential benefits and limitations of using these compounds against the burden of chronic diseases.

Mechanisms of estrogen effects on the endothelium: an overview.

In this review, we aim to provide an overview of the recent advances in understanding estrogen effects on the vascular endothelium. Epidemiological studies suggest the female sex hormone estrogen mediates the relative protection of premenopausal women against cardiovascular disease, compared with age-matched men. However, results from clinical trials of exogenous estrogen supplementation in postmenopausal women have been disappointing, generating much controversy about the role of estrogen and demonstrating the need for further research in this field. Here we have discussed the roles of different estrogen receptors (ERs) such as ERα, ERß, and G-protein coupled receptor 30; the complex genomic and nongenomic signalling pathways downstream to ER activation and the factors such as age, menopause, pregnancy, and diabetes that might alter estrogen responses. The common themes of this discussion are the complexity and diversity of endothelial estrogen responses and their modulation by 1 or more coexisting factors. Finally, we summarize the emerging therapeutic options including improved targeting of individual ERs and signalling pathways that might maximize the therapeutic potential of estrogenic compounds while minimizing their harmful side effects.

Egg-derived tri-peptide IRW exerts antihypertensive effects in spontaneously hypertensive rats.

BACKGROUND: There is a growing interest in using functional food components as therapy for cardiovascular diseases such as hypertension. We have previously characterized a tri-peptide IRW (Ile-Arg-Trp) from egg white protein ovotransferrin; this peptide showed anti-inflammatory, anti-oxidant and angiotensin converting enzyme (ACE) inhibitor properties in vitro. Given the pathogenic roles played by angiotensin, oxidative stress and inflammation in the spontaneously hypertensive rat (SHR), we tested the therapeutic potential of IRW in this well-established model of hypertension. METHODS AND RESULTS: 16-17 week old male SHRs were orally administered IRW at either a low dose (3 mg/Kg BW) or a high dose (15 mg/Kg BW) daily for 18 days. Blood pressure (BP) and heart rate were measured by telemetry. Animals were sacrificed at the end of the treatment for vascular function studies and measuring markers of inflammation. IRW treatment attenuated mean BP by ~10 mmHg and ~40 mmHg at the low- and high-dose groups respectively compared to untreated SHRs. Heart rate was not affected. Reduction in BP was accompanied by the restoration of diurnal variations in BP, preservation of nitric oxide dependent vasorelaxation, as well as reduction of plasma angiotensin II, other inflammatory markers and tissue fibrosis. CONCLUSION: Our results demonstrate anti-hypertensive effects of IRW in vivo likely mediated through ACE inhibition, endothelial nitric oxide synthase and anti-inflammatory properties.

Resveratrol prevents hypertension and cardiac hypertrophy in hypertensive rats and mice.

Resveratrol (RESV) is a polyphenol with pleiotropic effects that include reduction of oxidative stress and increased vascular nitric oxide (NO) production. However, whether or not RESV can prevent rises in blood pressure (BP) is controversial and remains to be firmly established. The purpose of this study was to determine whether RESV attenuates elevated BP and subsequent adaptive cardiac hypertrophy and to better understand the mechanisms involved. The spontaneously hypertensive rat (SHR) and the angiotensin (Ang)-II infused mouse were used as hypertensive models. Compared to a standard control diet, consumption of diets containing RESV by SHRs and Ang-II hypertensive mice, markedly prevented rises in systolic BP. In addition, flow-mediated vasodilation was significantly improved by RESV in SHRs. RESV also reduced serum and cardiac levels of the lipid peroxidation by-product, 4-hydroxy-2-nonenal in the hypertensive rodents and inhibited the production of superoxide in human-derived endothelial cells. Analysis of mesenteric arteries from SHRs and Ang-II infused mice demonstrated that RESV increased endothelial NO synthase (eNOS) phosphorylation by enhancing the LKB1/adenosine monophosphate (AMP)-activated protein kinase (AMPK) signal transduction pathway. Moreover, RESV reduced hypertrophic growth of the myocardium through reduced hemodynamic load and inhibition of the p70 S6 kinase pro-hypertrophic signaling cascade. Overall, we show that high dose RESV reduces oxidative stress, improves vascular function, attenuates high BP and prevents cardiac hypertrophy through the preservation of the LKB1-AMPK-eNOS signaling axis.

Structure and activity study of egg protein ovotransferrin derived peptides (IRW and IQW) on endothelial inflammatory response and oxidative stress.

Egg protein ovotransferrin derived peptides (IRW and IQW) can attenuate tumor necrosis factor (TNF) induced inflammatory responses and oxidative stress in endothelial cells. The present study investigates the structural requirements and molecular mechanisms underlying these events. Whereas IRW significantly inhibited TNF-induced up-regulation of intercellular cell adhesion molecule-I (ICAM-1) and vascular cell adhesion molecule-I (VCAM-1), IQW could inhibit only the up-regulation of ICAM-1. The anti-inflammatory effects of these peptides appeared to be mediated by the nuclear factor-κB (NF-κB) pathway, which was differentially regulated by IRW and IQW. Both IRW and IQW exhibited antioxidant effects as shown by reduction of TNF-induced superoxide generation. The structural integrity of these peptides was essential for their activities, because dipeptides or the combination of constituent amino acids did not exhibit the same effect. This study demonstrated the significance of the structural integrity of these two tripeptides in attenuating endothelial inflammation and oxidative stress, indicating their potential as nutraceuticals.