BENEFICIAL EFFECTS OF CHOLINE FROM EGGS ON THE BIOMARKERS OF HEART DISEASE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1013428
• Project Start Date: Oct 1, 2017
• Project End Date: Jan 4, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF CONNECTICUT
438 WHITNEY RD EXTENSION UNIT 1133
STORRS,CT 06269

Performing Department
Nutritional Sciences

Non Technical Summary
Egg consumption confers a broad range of health benefits associated with decreases on the biomarkers for heart disease risk including 1) increasing HDL cholesterol and HDL subfractions postulated to be involved in reverse cholesterol transport 2) increasing plasma lutein and zeaxanthin, carotenoids shown to decrease oxidative stress and inflammation and 3) providing choline, an essential nutrient which has anti-inflammatory effects and is important for liver health. However, despite the Dietary Guidelines recommendations released in January 2016, there is still uncertainty in the general public, who still perceive egg consumption to have adverse effects on health. This discrepancy has been further complicated due to concerns related to choline intake. Choline consumption has been shown to increase circulating levels of trimethylamine N-oxide (TMAO), a blood metabolite linked with increased heart disease risk. TMAO can be generated through conversion of dietary choline by intestinal bacteria to trimethylamine, which is absorbed and rapidly oxidized to TMAO by hepatic flavin-containing monooxygenases. Studies addressing TMAO formation and atherosclerosis have mainly used free choline, which is absorbed differently than phosphatidylcholine (PC), the major form of choline in eggs. Therefore, there is a critical need to conduct well-controlled clinical trials in healthy populations and those at risk for diabetes or heart disease to clearly establish any differences between choline intake either as a supplement (free choline) or from eggs (PC), relative to TMAO formation. In this project the effects of choline as a supplement or egg PC will be tested in healthy populations and in participants classified with metabolic syndrome. This well-controlled crossover study will provide key information on how plasma choline (protective nutrient) and plasma TMAO (biomarker of heart disease) are affected by the consumption of eggs when compared to a choline supplement in a randomized crossover design study. Other potential protective effects will be evaluated by comparing the effects of eggs (choline as phosphatidyl choline) versus choline supplementation on oxidative stress, low grade inflammation and dyslipidemias.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
701	3270	1010	100%

Knowledge Area
701 - Nutrient Composition of Food;

Subject Of Investigation
3270 - Eggs;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

eggs

choline

plasma tmao concentrations

inflammation

oxidative stress

plasma hdl cholesterol

reverse cholesterol transport

Goals / Objectives
The major goal of this project is to evaluate the effects of choline from eggs on plasma concentrations of choline and of trimethyl-N-amineoside, a marker of cardiovasular disease.General Hypotheses:Choline provided as PC from eggs will not be significantly converted into TMAO and therefore it will not increase the risk for heart disease in healthy subjects or in populations at risk for heart disease or diabetes, mainly subjects with metabolic syndrome.Egg intake will provide a number of benefits to these populations due to choline and other nutrients present in eggs, which will reduce the biomarkers for heart disease and diabetesThese two hypotheses will be translated into 5 major objectivesMeasure the chronic concentrations of plasma choline, trimethyalmine (TMA), TMAO and choline metabolites (e.g.,betaine) in plasma samples of diverse populations who will participate in clinical trials.Measure the postprandial effects of eggs versus choline supplementation on the post-prandial response to TMAOQuantify the expression of genes involved in HDL metabolism and those that are regulated by TMAO in peripheral blood mononuclear cells (PBMC) isolated from participantsDetermine the effects on plasma lipids, glucose, apolipoproteins and lipoprotein size and subfraction numberDetermine the effects of these interventions on HDL metabolism and on plasma antioxidants

Project Methods
Experimental Design We will recruit 30 healthy individuals (aged 18-30 years) with plasma cholesterol < 240 mg/dL, and body mass index (BMI) between 18.5-30 kg/m2. The proposed population will consist of 15 men and 15 women to account for variability based on gender. Subjects will be randomly allocated to consume either 3 eggs per day or the equivalent amount of choline in a supplement for 4 weeks (390 mg of choline). Based on the standard deviation from our previous studies and using a Z value of 1.96 (95% confidence interval), we needed to recruit 28 patients to detect differences in plasma choline following egg consumption. We are recruiting 30 subjects to allow for attrition. We are using 3 eggs to ensure that we have relatively high concentrations of choline similar to what is given in supplement form. Following the first dietary intervention, all subjects will undergo a 4 week washout (to ensure that potential formation of TMAO with choline is washed out). After the washout, they will be allocated to the alternate treatment. Fasting plasma and PBMCs will be isolated at each time point when blood is drawn. Similar protocol will be used to recruit subjects with metabolic syndrome and the inclusion/exclusion criteria will be similar with the exception of BMI, which will be between 25-35 kg/m2. Other exclusion criteria will be blood glucoses > 126 mg/dL and taking of glucose lowering medications.For both of these populations, a post-prandial study, will also be conducted in which a subset of 10 subjects will be given either 3 eggs or 390 mg of choline tartrate and plasma samples will be taken at baseline and every hour during 6 hours. Plasma choline, TMA and TMAO will also be measured at each time point to determine increases of both choline and TMAO after ingestion.Diet Monitoring: Subjects will be asked to provide 5-d dietary records (3 wk days and two wk end days) after the end of each 4-wk dietary period. Diet will be analyzed by using the Nutritional Data System for Research 2014 (Minneapolis, MN). Dietary compliance will be assessed by monitoring daily intake of eggs and choline supplement in sheets provided to participants. In addition, subjects will come every 2 weeks to pick up their eggs or supplement to monitor compliance.Blood Processing: Eighty mL of fasting blood will be collected from each subject at the end of each dietary treatment into EDTA Vacutainer tubes (70 mL) or serum separator tubes (10 mL). Blood will be centrifuged at 2,000 x g for 20 min to separate plasma that will be aliquoted into 500 μL tubes for the different measurements.Techniques TMAO: Stable isotope dilution liquid chromatography with on-line tandem mass spectrometry (LC/MS/MS) will be used to quantify choline, TMA, and TMAO levels in plasma according to Wang et al..Isolation of Peripheral Blood Mononuclear Cells (PBMC): Fasting blood (50 mL) will be collected into EDTA Vacutainer tubes at the end of each dietary period. PBMC will be isolated by density gradient centrifugation using Ficoll Paque PLUS (GE Healthcare, Pittsburgh, PA). PBMC buffy coats will be collected, washed twice with PBS, resuspended, and kept at -80°C until mRNA isolation (18).Gene expression; ATP binding cassette transporter A1 (ABCA1), ATP binding cassette subfamily G1 (ABCG), scavenger receptor B1 (SR-B1) (involved in HDL metabolism) and cluster of differentiation (CD)-36 and macrophage scavenger receptor 1 (SR-A) (upregulated by TMAO) and inflammatory genes tumor necrosis factor α (TNFα), interleukin (IL)-1β, IL-6, toll-like receptor 4 (TLR4) will be analyzed by extracting RNA from isolated PBMCs using the TRIzol method. cDNA will be reverse transcribed from RNA and mRNA expression will be measured by real-time qRT-PCR using SYBR Green and a CFX96 real-time PCR detection system (Bio-Rad). Fold expression relative to an endogenous control gene will be calculated as 2−ΔΔCt..Plasma lipids, Glucose, C reactive Protein (CRP) and Liver Enzymes: Our laboratory has been part of the Lipid Standardization Program of the Centers for Disease Control-National Heart, Lung and Blood Institute (CDC-NHLBI) since 1989. Plasma total cholesterol, LDL-C and HDL-C, triglycerides, as well as liver enzymes, glucose and C-reactive protein will be measured by utilizing a Cobas Analyzer c 111 (Roche Diagnostics).Plasma Apolipoproteins: We will measure plasma apolipoproteins utilizing Luminex x MAP multiplexing on a MAGPIX instrument (EMD Millipore). We quantify apolipoproteins A-I, A-II, B, C-II, C-III and E simultaneously. These apolipoproteins are involved in the metabolism of HDL and atherogenic lipoproteins. We have demonstrated highly reproducible results using this method.Determination of Size and Number of VLDL, LDL and HDL Subfractions: Lipoprotein subclass profiles will be measured by proton NMR spectroscopy. The method uses characteristic signals broadcast by lipoprotein subclasses of different size including large, medium and small VLDL, large, medium, small and very small LDL and large, medium and small HDL. This method provides key information regarding atherogenicity of lipoproteins.LCAT and CETP Activities: We will measure LCAT and CETP activity as a way to assess changes in RCT due to increased egg intake. Plasma LCAT and CETP activities will be determined by using assay kits (Roar Biomedical). Activity will be expressed as mmol/L/h. The intra-assay variability in our laboratory is 2.2%.Determination of Inflammatory MarkersTNF-α, IL-6, IL-10, and the adhesive molecules ICAM and VCAM will be measured by Multiplex Technology.HDL-Serum Amyloid A (SAA): SAA concentration will be examined in plasma by sandwich enzyme-linked immunosorbent assay (ELISA). The major proportion of plasma SAA (~95%) is transported by HDL particles. HDL-SAA concentration is considered a marker of pro-inflammatory HDL, whereby HDL loses its ability to prevent endothelial cell inflammation.HDL antioxidant assays: Serum will be measured for PON1 activity towards phenyl acetate as described by Rock et al.with substrate hydrolysis measured using a spectrophotometer at 270 nm every 20 seconds for 3 minutes (25°C).HDL antioxidant capacity: HDL fractions will be isolated by ultracentrifugation (UC) and tested for their ability to prevent the spontaneous oxidation of the fluorescent probe, dihydrorhodamine 123 (DHR) UC-isolated HDL (2.5 µg cholesterol) will be incubated with 10 µM DHR (Life Technologies) and 150 µM of 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) (Sigma-Aldrich) to initiate oxidation. The oxidation of DHR will be monitored every 2 min for 1 hour at excitation wavelength of 500 nm and emission wavelength of 536 nm.HDL anti-inflammatory assays: The ability of HDL to protect against endothelial cell inflammation will be examined. Confluent human umbilical vascular endothelial cells (ATCC) will be incubated with UC-isolated HDL in 10% FBS-containing media for 16 hours at 37°C in 5% CO2. Cells will be incubated for an additional 5 hours in basal or stimulation media containing recombinant TNFα (0.1 ng/mL). Cell media will be collected and the amount of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) protein secreted by cells will be determined by ELISA. Cells will be washed and total RNA will be isolated from cells using TRIzol reagent (Life Technologies).

Progress 10/01/17 to 01/04/21

Outputs
Target Audience:Target audiences included attendees at scienitic meetings, and faculty and students attending seminars or defense presentations Changes/Problems:This project was terminiated after three years due to a change in the process of allocating Capacity Funds. What opportunities for training and professional development has the project provided?Training of one PhD student and one Master's student plus 4 honor's thesis How have the results been disseminated to communities of interest?The results have been presented at scientific meetings and have been published What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? All the goals were accomplished. The project has been finished. The project goals for the healthy population were completely covered. Studies measure the postprandial effects of eggs versus choline supplementation on the post-prandial response to TMAO 3, quantifed the expression of genes involved in HDL metabolism and those that are regulated by TMAO in peripheral bloodmononuclear cells (PBMC) isolated from participants, determined the effects on plasma lipids, glucose, apolipoproteins and lipoprotein size and subfraction number, and determined the effects of these interventions on HDL metabolism and on plasma antioxidants. The last year of the proejct studies involved the metabolic syndrome population to evaluate the effects of choline from eggs vs choline from a supplement (cholinebitartrate) in changing the microbiota and affecting inflammation and insulin resistance in this population at risk for heartdisease and diabetes. The intervention part of the study included twenty three subjects; only 20 were needed to observe statistical differences in the main variables. Data was analyzed and found there were no significant differences in plasma lipids or glucose duringthe egg compared to the choline supplement period. This study data suggests that egg intake contributes to choline availability in plasma when comparing to choline bitartrate supplementation, while the dietary cholesterol regulates the endogenous synthesis of cholesterol without negatively impacting risk for CVD in young, healthy individuals.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Dibella M, Thomas MS, Alyousef H, Millar C, Blesso C, Malysheva O, Caudill MA, Fernandez ML. Choline Intake as Supplement or as A Component of Eggs Increases Plasma Choline and Reduces Interleukin-6 without Modifying Plasma Cholesterol in Participants with Metabolic Syndrome, Nutrients 2020;12: 3120
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Thomas MS, Fernandez ML. Trimethylamine N-Oxide, diet and cardiovascular disease. Current Atherosclerosis Rep. 2020 (in press)
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Khaligi M, Soltani S, Kolahdouz-Mohammadi, Fernandez ML, Varse Fatemeh, Shidfar F. The responses of different dosages of egg consumption on blood lipid profile. An Updated systematic review and meta-anlaysis of randomized clinical trials. J Food Biochem. 2020

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The target audiences this reporting period include scientists, students and lay people. Data from this project was presented at the ASN meeting in Baltimore on June, 2019; Dr. Fernandez gave three talks in Argentina, one to the Research Dietitians Association, another to the hospital (Nurses and doctors) and a third one to students from the University of Salta, Salta, Argentina in May 2019. In addition, data was presented to the International Congress in Barcelona, Spain in September 2019. Changes/Problems:No major problems were identified What opportunities for training and professional development has the project provided?This project provided research development training and was the subject of the PhD dissertation for Bruno Lemos who graduated in January of 2019. How have the results been disseminated to communities of interest?Findings from this project were presented to scientists at the ASN Nutrition meeting, and to the community of research dietitians, doctors, nurses and students in Argentina. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The project goals for the healthy population were completely covered. What we have been working on in the past year is the metabolic syndrome population to evaluate the effects of choline from eggs vs choline from a supplement (choline bitartrate) in changing the microbiota and affecting inflammation and insulin resistance in this population at risk for heart disease and diabetes. At this point, we finished the intervention part of the study. Twenty three subject completed the intervention last December; we needed only 20 to observe statistical differences in the main variables. We are in the process of doing all analsyis. We have finished plasma lipids and glucose. There were no significant differences in plasma lipids or glucose during the egg compared to the choline supplement period. We are wroking in the measurmetns of other biomarkers plasma choline and TMAO as well as plasma insulin and inflammatory markers. We expect to present these data in the ASN meeting in Seattle, WA on May 30 to June 2.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: DiMarco D, Fernandez ML. Differences in response to egg-derived dietary cholesterol result in distinct lipoprotein profiles while plasma concnetrations of carotenoids and choline are not affected in a young healthy population. J Agricult Food Res 2019 (in press).
• Type: Book Chapters Status: Published Year Published: 2019 Citation: Fernandez ML, Lemos BS. Eggs is a Natural Functional Food in Eggs as Functional Foods and Nutraceuticals for Human Health. The Royal Society of Chemistry 2019: pages 22-31
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Murillo AG, Hu S, Fernandez ML. Zeaxanthin: Metabolism, Properties and Antioxidant Protection of Eyes, Heart, Liver and Skin. Antioxidants 2019; 11:8 doi: 10.3390/antiox8090390
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Fernandez ML. Commentary: Small HDL particles are associated with gestational diabetes, a potential early identification tool. J. Nutr. 2019
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Serra-Majemm L, Fernandez ML. Nutrients 2009-2019: The present and the future of Nutrition. Nutrients 2019:11,88; doi:10.3390/nu11010088

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audiences this reporting period were attendees at scientific meetings both National and International, egg producers and graduate students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two papers have been submitted to publication from the collected data. A PhD Dissertation will be the result of the collected data How have the results been disseminated to communities of interest?Results have been dessiminated 1. A Meeting in Des. Moins, Iowa where results were presented to the Egg Producers 2. A meeting in Cartagena, Colombia in a Pediatritians Congress What do you plan to do during the next reporting period to accomplish the goals?More asssays are being done to do finish the evaluation of the antioxidant capacity of HDL

Impacts
What was accomplished under these goals? Goals 1-4 have been accomplished and we are currently working on goal 5 The effects of egg intake in comparison to choline bitartrate supplement was studied in a young, healthy population. Thirty participants were enrolled for a 13-week intervention. After a 2-week washout, subjects were randomized to consume either 3 eggs/day or take choline bitartrate (~400mg choline for both treatments) for 4-weeks each. After a 3-week washout period, they were allocated to the alternate treatment. Dietary records, plasma lipids, apolipoproteins (Apo) concentrations and gene expression of regulatory genes of cholesterol homeostasis, in isolated peripheral blood mononuclear cells, were assessed at the end of each intervention. Following the consumption of eggs, saturated and monounsaturated fat intake was higher when compared to the choline period. In addition, higher total cholesterol, HDL and LDL cholesterol were observed with egg consumption (P < 0.01), while no change was seen in LDL/HDL ratio, a key marker of heart disease risk. Compared to choline supplementation, intake of eggs resulted in higher concentrations of plasma Apo-AI and Apo-E with no changes in apo B. 3-hydroxy-3-methyl glutaryl CoA reductase and sSterol regulatory element binding protein-2 expression were lower with egg consumption (P < 0.05). Dietary cholesterol from eggs appears to regulate endogenous synthesis of cholesterol in a way that the LDL/HDL ratio is maintained In addition, compared to the choline supplement, intake of total fat, cholesterol, selenium and vitamin E were higher (p < 0.05), whereas carbohydrate intake was lower (p < 0.001) with consumption of 3 eggs/d. Fasting plasma choline increased 20% (p = 0.023) with egg intake, while no changes were observed with choline supplementation. Plasma TMAO levels were not different between dietary treatments or compared to baseline. Dietary choline appears to be more bioavailable via egg consumption when compared to a choline supplement. Plasma TMAO concentrations were not affected in healthy subjects after 4 weeks of taking ~400 mg/d choline either via eggs or choline supplementation.

Publications

• Type: Books Status: Published Year Published: 2018 Citation: Egg Consumption and Human Health. published by MDPI, Edited by Maria Luz Fernandez
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Fernandez ML, Commentary: High fat breakfast and fat oxidation in older adults J. Nutr. 2018 (in press)
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Vergara-Jimenez M, Almatrafi MM, Fernandez ML.. Bioactive Components in Moringa Oeifera leaves protect against chronic Disease. Antioxidants 2017; 6,: 91
• Type: Journal Articles Status: Published Year Published: 2018 Citation: 3. Missimer A, Fernandez ML, DiMarco DM, Norris GH, Blesso CN, Murillo AG, Vergara-Jimenez M, Lemos BS, Medina-Vera I,k Malysheva OV, Caudil MA. Compared to an oatmeal breakfast, two eggs/day increased plasma carotenoids and choline withouth increasing trimytheyl amine-oxide concentations. J. Am Coll Nutr 2018;37:148-148
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Almatrafi MM, Vergara-Jimenez M, Smyth JA, Medina-Vera I, Fernandez ML. Moringa olifeira leaves do not alter adipose tissue colesterol accumulation or inflammation in guinea pigs fed a hypercholesterolemic diet. EC Nutrition 2017;18:1330
• Type: Journal Articles Status: Published Year Published: 2017 Citation: DiMarco D, Norris GG, Blesso CN, Fernandez ML. Intake of up to 3 eggs per day does not impact expression of genes related to cholesterol homeostasis, uptake and transport in a healthy population. Prensa Med. Argentina, 2017 103:1
• Type: Journal Articles Status: Published Year Published: 2017 Citation: DiMarco DM, Norris GH, Millar C, Blesso C, Fernandez ML. Intake of up to 3 eggs per day is associated with changes in HDL function and increased plasma antioxidants in healthy young adults. J. Nutr. 2017; 147:323-339
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Fernandez ML. Eggs and Health, Special Issue, Editorial, Nutrients 2016; 2:8(12).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Murillo AG, Almatrafi M, Vergara-Jimenez M, Fernandez ML. 2017. Moringa Leaf poder modulates reverse cholesterol transport withouth changing plasma lipids in guinea pigs. FASEB J A647.2.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Almatrafi M, Vergara-Jimenez M, Murillo AG, Norris GN, Blesso CN and Fernandez ML. 2017. Moringa leaf powder attenuates hepatic steatosis and decreases inflammation by altering the expression of genes involved in lipid synthesis. FASEB J A793.17.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Missimer A, DiMarco DM, Murillo AG, Millar CL, Blesso CN, Fernandez ML. 2017. Consumption of 2 eggs per day as compared to an oatmeal breakfast increases plasma carotenoids and markers associated with reverse cholesterol transport in young, healthy individuals. FASEB J A. 431.7 (oral).
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Lemos BS, DiMarco DM, Missimer A, Murillo AG, Malysehva OV, Caudill MA, Blesso CN, Fernandez ML. 2017. Consumption of up to three eggs per day increases Dietary cholesterol and choline while plasma LDL cholesterol and trimethylamin N-oxide concentrations are not increased in a young healthy population. FASEB J A447.3 (oral).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: DiMarco DM, Norris GH, Millar CL, Blesso CN, Fernandez ML. 2017. Intake of up to three eggs per day is associated with improvements in markers of HDL function and antioxidant capacity in a young healthy population. FASEB J A:137.5 (oral).