Cholesterol in Chicken Eggs: Still a Dietary Concern for Some

Chapter 18

Cholesterol in Chicken Eggs: Still a Dietary Concern for Some Robert G. Elkin Department of Animal Science, The Pennsylvania State University, University Park, PA, United States

INTRODUCTION In his monograph “The Cholesterol Wars,” Daniel Steinberg analyzes the controversy surrounding the “lipid hypothesis” that occurred during the latter half of the 20th century (Steinberg, 2007). The “lipid hypothesis” postulates that hypercholesterolemia or dyslipidemia [elevated circulating low-density lipoprotein (LDL)-cholesterol or low circulating highdensity lipoprotein (HDL)-cholesterol or both] is a major causative factor—but not the only factor—in atherosclerosis and coronary heart disease. Steinberg (2007) also carefully pointed out that the hypothesis relates to blood lipids and not to dietary lipids, although dietary lipids can influence the levels of blood lipids, and that many other factors play important roles, including individual variability in the response to dietary changes (which will be discussed in more detail later). He also remarked that: “The importance of dietary cholesterol as a determinant of blood cholesterol levels has probably been overstated; certainly saturated fat intake is quantitatively more important. However, cholesterol intake is relevant.” Although still not universally accepted, there is overwhelming evidence that elevated levels of plasma LDL-cholesterol are associated with an increased risk of cardiovascular disease (CVD; Stein, 2009; Goldstein and Brown, 2015). At the time that this chapter was being written, the United States Department of Health and Human Services and the United States Department of Agriculture were mulling over a recommendation from the 2015 Dietary Guidelines Advisory Committee (DGAC) to drop a longstanding recommendation that Americans restrict their dietary intake of cholesterol (Dietary Guidelines Advisory Committee, 2015). The DGAC stated that “available evidence shows no appreciable relationship between consumption of dietary cholesterol and serum cholesterol.” This statement likely holds for the majority of healthy individuals in a population, but there are many published reports that demonstrate that the response to changes in dietary cholesterol in humans is heterogeneous. In addition to hyperresponders who, as the name implies, exhibit a hyperresponse in plasma LDL-cholesterol to dietary cholesterol, and for others at risk of CVD, including diabetics, this DGAC statement is in reality a half-truth. Hyperresponders account for approximately 25% (or more) of the normal population (Katan et al., 1986; McNamara et al., 1987; McNamara, 2000; Schaefer, 2002; Herron et al., 2002, 2003, 2006; Barona and Fernandez, 2012), whereas the 2014 National Diabetes Statistics Report stated that 29.1 million Americans (9.3% of the US population) have diagnosed or undiagnosed diabetes (Centers for Disease Control and Prevention, 2015). The report also mentions that from 2009 to 2012, of adults aged 18 years or older with diagnosed diabetes, 65% had circulating LDLcholesterol levels ≥ 100 mg/dL or used cholesterol-lowering medications. According to the National Heart, Lung, and Blood Institute (http://www.nhlbi.nih.gov/health/resources/heart/heart-cholesterol-hbc-what-html), for most individuals, a circulating LDL-cholesterol level < 100 mg/dL is considered optimal, although some cardiologists and researchers opine that lower is even better in the context of CVD risk. Despite evidence demonstrating a link between dietary cholesterol intake and serum cholesterol levels, particularly in high-risk populations, such as hyperresponders and diabetics, the new 2015–2020 Dietary Guidelines for Americans (United States Department of Agriculture and the United States Department of Health and Human Services, 2016) subsequently dropped the long-standing recommendation to limit the consumption of dietary cholesterol to 300 mg/day. Paradoxically, the new Guidelines state “…individuals should eat as little dietary cholesterol as possible while consuming a healthy eating pattern.” As a result, in January 2016, the Physicians Committee for Responsible Medicine filed a suit in the Egg Innovations and Strategies for Improvements. http://dx.doi.org/10.1016/B978-0-12-800879-9.00018-4 Copyright © 2017 Elsevier Inc. All rights reserved.

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United States District Court for the Northern District of California (Physicians Committee for Responsible Medicine, 2016) contending that the decision to no longer recommend limiting dietary cholesterol intake to 300 mg/day was the result of improper influence from research funded by the United States Department of Agriculture’s egg promotion program and from funds received from the egg industry. Eggs are among the most cholesterol-rich foods consumed by man, and concerns (both historical and current) about egg consumption and CVD risk center on their cholesterol content (Eckel, 2008). Although current dogma suggests that egg consumption is not associated with CVD risk in healthy populations, Zampelas (2012) stated that the evidence is inconclusive when high-risk populations are considered. Thus, it is the opinion of the author that the production of shell eggs with a markedly reduced cholesterol content of greater than 50% (1) remains a viable research goal; (2) would be a desirable dietary option for the millions of Americans who need to restrict their cholesterol intake but wish to include whole eggs or egg yolks in their diet; and (3) would command a premium in the marketplace. This chapter will initially discuss the link between consumption of cholesterol from eggs and the risk of CVD; it will conclude with an overview of approaches to reducing the cholesterol content of shell eggs.

META-ANALYSES VERSUS OBSERVATIONAL STUDIES: GROUPS VERSUS INDIVIDUALS Before embarking on a discussion of reports that either substantiate or refute the role of eggs in CVD, it is important to mention the caveat of Chang (2013) who stated: “One common concern with meta-analyses is that they report average estimates of effect that may or may not apply to all individuals or situations.” An extreme example of how data from groups versus individuals can be misinterpreted is discussed by Maurage et al. (2013), who critiqued a study relating chocolate consumption by country to the number of Nobel laureates at the national level. They mentioned that such a misinterpretation problem is known as “ecological inference fallacy,” where conclusion about the behavior of individuals is drawn from aggregated behaviors, with no surety that relationships observed at the group level necessarily hold for individuals. On the other hand, results from randomized controlled trials (RCTs), and syntheses of RCTs, may lack the power to identify differences over long time periods, and typically lack the sample size to confirm findings obtained through meta-analyses (Chang, 2013). Thus, neither meta-analyses of RCTs, nor RCTs themselves, should be viewed as providing the unequivocal answer to a question when human subjects are involved. However, it is the opinion of the author that any study that only reports treatment (group) averages in response to changes in dietary cholesterol is potentially misleading to the reader, because this response in humans has been clearly shown to be heterogeneous (Ahrens, 1984a,b; Sacks et al., 1984; Katan et al., 1986; McNamara et al., 1987; Schaefer, 2002; Herron et al., 2002, 2003, 2006). Furthermore, as pointed out by Ahrens (1984a): “Any heterogeneity of responsiveness to diet and/or drug interventions will be glossed over if data are presented as averages. People are not pure-bred laboratory animals.”

CHOLESTEROL IN CHICKEN EGGS: WHY IT SHOULD STILL BE A TARGET FOR REDUCTION Based on a growing body of epidemiological research, as well as a number of prospective population-based studies that scrupulously accounted for other dietary variables (such as saturated fatty acids), a cadre of researchers and clinicians shared the opinion that there is a very minimal association (if any) between cholesterol intake in general or egg intake in particular, and the risk of CVD (reviewed by Gray and Griffin, 2009; Fernandez, 2010; Kanter et al., 2012). Although this may be true for healthy individuals (including compensators or hyporesponders), there is ample published evidence to suggest that this in fact does not hold true for certain subgroups of the population (noncompensators or hyperresponders, diabetics, etc.), which represent millions of Americans. For example, 38% of 40 healthy male subjects (mean age 32.6 years) and 39% of 51 healthy premenopausal women (mean age 29.5 years) were classified as hyperresponders (Herron et al., 2002, 2003, 2006) and exhibited significant increases in plasma LDL-cholesterol and elevated ratios of LDL-cholesterol/HDL-cholesterol in response to a dietary cholesterol challenge (daily addition of the liquid equivalent of 3 eggs totaling approximately 640 mg cholesterol, Table 18.1). In contrast, McNamara (2000), Fernandez (2010), and Barona and Fernandez (2012) cite other studies that report equal elevations in both LDL-cholesterol and HDL-cholesterol in hyperresponders such that the LDL/HDL ratio was unchanged. In addition, Kanter et al. (2012) and Barona and Fernandez (2012) review work that suggests that cholesterol ingestion results in the formation of larger LDL and HDL particles, implying that the former are less atherogenic and that the latter are more effective at reverse cholesterol transport. Yet, ironically, the reports of Herron et al. (2002, 2003, 2006), Fernandez (2010), and Barona and Fernandez (2012) all originated from the same laboratory. Moreover, in a review paper, McNamara (2000) failed to cite his own seminal study (McNamara et al., 1987) that clearly demonstrated the existence of a heterogeneity of cholesterol homeostasis in humans, while concluding that “…dietary cholesterol has little effect on the plasma LDL:HDL ratio” and that “…egg consumption and CHD rates uniformly indicate

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TABLE 18.1 Plasma Cholesterol Response of Human Female and Male Hyperresponders and Hyporesponders during Periods of Consumption of the Liquid Equivalent of Three Whole Eggs (640 mg cholesterol/day) or a Cholesterol-Free Egg Substitute (CFES) Added to Their Diets Plasma Cholesterol Total (mg/dL)

Low-Density Lipoprotein (mg/dL)

High-Density Lipoprotein (mg/dL)

LDL/HDL

Egg

—

120.6

68.0

1.80

CFES

—

96.7

60.7

1.63

0.0001

0.001

NS

Study

Sex

Group

1a

Female (n = 20)

Hyperresponders

P-value 1

Female (n = 31)

Hyporesponders Egg

—

102.8

60.3

1.62

CFES

—

92.8

59.2

1.63

NS

NS

NS

P-value 2b

2

c

3

3

3

3

Male (n = 15)

Male (n = 25)

Female (n = 20)

Female (n = 31)

Male (n = 15)

Male (n = 25)

Hyperresponders Egg

188.7

111.0

49.9

2.33

CFES

159.7

85.4

46.0

1.91

P-value

0.0001

0.0001

0.05

0.05

Egg

167.4

94.3

47.9

2.07

CFES

169.3

95.9

48.3

2.08

P-value

NS

NS

NS

NS

Egg

199.2

115.8

68.0

1.70

CFES

171.8

94.6

60.5

1.56

P-value

0.001

0.0001

0.001

Egg

169.3

91.9

61.4

1.49

CFES

171.1

92.8

60.3

1.53

P-value

NS

NS

NS

Egg

187.3

110.3

49.4

2.23

CFES

158.3

85.5

46.4

1.84

P-value

0.0001

0.0001

0.05

Egg

167.6

94.5

47.8

1.97

CFES

166.7

96.0

48.5

1.98

P-value

NS

NS

NS

Hyporesponders

Hyperresponders

Hyporesponders

Hyperresponders

Hyporesponders

HDL, High-density lipoprotein; LDL, low-density lipoprotein. a Herron, K.L., Vega-Lopez, S., Conde, K., Ramjiganesh, T., Roy, S., Shachter, N.S., Fernandez, M.L., 2002. Pre-menopausal women, classified as hypo- or hyper-responders, do not alter their LDL/HDL ratio following a high dietary cholesterol challenge. J. Am. Coll. Nutr. 21, 250–258. Reprinted by permission of the American College of Nutrition (http://www.americancollegeofnutrition.org). Data adapted from Table 5. Plasma total cholesterol values were not reported. b Herron, K.L., Vega-Lopez, S., Conde, K., Ramjiganesh, T., Shachter, N.S., Fernandez, M.L., 2003. Men classified as hypo- or hyperresponders to dietary cholesterol feeding exhibit differences in lipoprotein metabolism. J. Nutr. 133, 1036–1042. Data adapted from Table 4. c Herron, K.L., Lofgren, I.E., Adiconis, X., Ordovas, J.M., Fernandez, M.L., 2006. Associations between plasma lipid parameters and APOC3 and APOA4 genotypes in a healthy population are independent of dietary cholesterol intake. Atherosclerosis 184, 113–120. Data adapted from Table 2. The plasma LDLcholesterol/HDL-cholesterol ratios were not reported and were calculated by the author.

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a null relationship” (CHD, coronary heart disease). In the McNamara et al. (1987) study, the cholesterol challenge was provided by instructing each patient to add three large eggs daily to their original diet. It is also known that adding cholesterol to a cholesterol-free diet raises the blood level in humans, but when added to an unrestricted diet it has a minimal effect (Keys, 1991). Steinberg (2007) reviewed this topic and suggested that this is probably why some researchers have observed no effect of adding eggs to a basal diet that contained more than 300 mg cholesterol, whereas others found that adding eggs had a significant plasma LDL-cholesterol-elevating effect when the basal diet was low in cholesterol (e.g., the study of Sacks et al., 1984). Berger et al. (2015) recently examined the effects of dietary cholesterol on CVD risk in healthy adults by using systematic review and meta-analysis. Based on the results of 40 publications (19 from 17 prospective cohort studies and 21 from 19 dietary intervention trials), the authors reported that dietary cholesterol increased (statistically significantly) total serum cholesterol, LDL-cholesterol, and the LDL-cholesterol/ HDL-cholesterol ratio. However, when dietary intervention dose exceeded 900 mg/day, increases in serum total- and LDLcholesterol were no longer statistically significant. Assessments of LDL and HDL subparticles were not included in the study. Despite these findings, Berger et al. (2015) concluded that the reviewed studies were heterogeneous and lacked the methodologic rigor to draw any conclusions regarding the effects of dietary cholesterol on CVD risk. In contrast, others have reported that regular consumption of egg yolk should be avoided by persons at risk of CVD (Spence et al., 2010, 2012), including diabetics (Hu et al., 1999; Qureshi et al., 2007; Lecerf and de Lorgeril, 2011; Rong et al., 2013), and that increasing dietary cholesterol results in elevated plasma cholesterol concentrations and is associated with an increased risk of coronary heart disease (reviewed by Schaefer, 2002). In an interesting cohort study, Djoussé and Gaziano (2008) found that Harvardeducated male physicians who ate ≥1 egg per day had a greater incidence of all-cause mortality, and that egg consumption was more strongly related to mortality if these health care professionals were diabetic.

OTHER EVIDENCE OF INTER-INDIVIDUAL VARIABILITY IN CHOLESTEROL HOMEOSTASIS IN HUMANS Cholesterol absorption rates in humans vary widely, ranging from 29% to 80%, with a mean of 56% (Bosner et al., 1999). In addition, people also differ in how they catabolize cholesterol as evidenced by the infamous “Egg Man.” In a report that garnered national headlines, Kern (1991) described an 88-year-old man who ate 25 eggs per day (approximately 5000 mg of cholesterol) for at least 15 years (and possibly longer) and maintained normal plasma cholesterol levels (total cholesterol, 200 mg/dL; LDL-cholesterol, 142 mg/dL; and HDL-cholesterol, 45 mg/dL; LDL-cholesterol/HDL-cholesterol ratio was 3.15). The patient also was almost completely free from clinically important atherosclerosis and its complications, as well as from gall bladder disease. Kern (1991) reported that the patient had extremely efficient compensatory mechanisms in that he absorbed only 18% of the dietary cholesterol, had a moderately reduced rate of cholesterol biosynthesis, likely had an increased rate of biliary cholesterol secretion, and had greatly increased bile acid biosynthesis. The latter is a major pathway of cholesterol elimination (Jones and Heuman, 1991). Cholesterol metabolism is a complex process involving multiple metabolic pathways with many points of regulation that are under genetic and metabolic control (Goldstein and Brown, 2015; Kapourchali et al., 2015). According to Jones and Heuman (1991), it therefore is not surprising that individual responses to dietary cholesterol are so heterogeneous and, in this context, the “Egg Man” represents the most extreme example of a compensator or hyporesponder. Interindividual variability in blood cholesterol levels in response to dietary plant sterols and stanols has also been recently reported (Jones, 2015). Unfortunately, as is the case with the heterogeneity of the response to dietary cholesterol, there are presently no biomarkers or genetic tests available to guide health care professionals in identifying hyperresponders. Thus, the only way to determine if one is a hyporesponder or a hyperresponder is to submit to a clinical trial. It is hoped that nutrigenetic differences across genes associated with cholesterol trafficking pathways eventually will allow one to predict how an individual will respond to dietary interventions (Jones, 2015). Until then, individualized dietary strategies for health optimization of hyperresponders remain elusive. In this context, the recommendation of the DCAG (2015) that Americans no longer restrict their dietary intake of cholesterol sends the wrong message to millions of people who should.

CHOLESTEROL CONTENT HAS BEEN DECREASING IN EGGS FROM AMERICAN FLOCKS Over the past 40+ years, the cholesterol content of eggs laid by American flocks has been decreasing. Feeley et al. (1972) reported the results of a study to update and expand the data on the cholesterol content of foods that appeared in the 1963 version of United States Department of Agriculture Handbook 8 (Composition of Foods—Raw, Processed, Prepared). They found that a large egg (50-g edible portion) contained 252 mg of cholesterol. In an extensive reevaluation of the nutrient content of eggs, Cotterill et al. (1977) determined that a 61-g egg (53-g edible portion) contained 260 mg of cholesterol.

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Almost three decades later, the cholesterol content of a large raw egg had decreased to 213 mg per 50 g edible portion (United States Department of Agriculture, 1989). Following a nationwide sampling of whole large eggs by the United States Department of Agriculture in 2010, Exler et al. (2013) determined that the cholesterol content had decreased further to 185 mg per 50 g edible portion. Although Exler et al. (2013) attributed this change to alterations of the hens’ diets, it is more likely that the increased rate of egg production in White Leghorn hens from 2000 to 2010, due to genetic advances, was responsible. It is well established that an inverse relationship exists between egg yolk cholesterol content and the rate of egg production (reviewed by Elkin, 2006). Egg production (as number of eggs per hen to 60 weeks of ge) of White Leghorn hens, the predominant strain used in the United States, has increased from approximately 165 to 228 to 265 from 1971 to 2001 to 2011, respectively (N. O’Sullivan, Hy-Line International, Inc., personal communication, 2012). Because the rate of egg production is inversely related to both yolk weight and yolk cholesterol concentration (Hall and McKay, 1993), and assuming that the White Leghorn hen’s ability to hepatically synthesize cholesterol has not risen proportionately (i.e., by 60%) from 1971 to 2011, then it would be expected that as egg production rates have increased, the eggs laid had comparatively smaller yolks and contained less total cholesterol.

EGG CHOLESTEROL CONTENTS: PERSPECTIVES ON ANALYTICAL TECHNIQUES AND REPORTING DATA Before reviewing the various experimental approaches to egg cholesterol reduction, mention must be made of two shortcomings that pervade the literature and were the subject of a guest editorial by the author in Poultry Science. Elkin (2009) discussed the considerable interlaboratory variation in reported yolk cholesterol contents and stressed the need for researchers to validate their methods by analyzing a standard reference material such as the Standard Reference Material (SRM) 1845 from the National Institute of Standards and Technology (now SRM 1845a; see https://www-s.nist.gov/srmors/view_ detail.cfm?srm=1845A). An additional concern is that some authors report egg cholesterol contents only on a per gram of yolk basis and in many cases also fail to report yolk weights, thus, the cholesterol content on a per yolk (i.e., per egg) basis cannot be determined. This lack of information is problematic because significant differences between treatments for relative cholesterol contents (per gram of yolk basis) may not translate into significant differences between treatments for total yolk (egg) cholesterol contents if yolk weights or yolk lipid contents or both differ between treatments. In addition, reporting cholesterol content on a whole yolk (egg) basis is of practical significance because most eggs (about 69%) in the United States are consumed as shell eggs versus processed egg products (United States Department of Agriculture Economic Research Service, 2014).

EXPERIMENTAL APPROACHES TO EGG CHOLESTEROL REDUCTION In the 1970s, when the “Cholesterol Wars” were being fought amongst the members of the biomedical research community, poultry scientists began to turn their attention toward trying to reduce the cholesterol content of eggs, whose consumption had been implicated as a contributing factor to CVD. At that time, it was known that the main yolk precursor macromolecules, very low–density lipoprotein (VLDL), and vitellogenin (VTG), were hepatically synthesized by the hen under the influence of estrogen, secreted into the bloodstream, and transported into growing oocytes (Burley and Vadehra, 1989). Very low-density lipoprotein and VTG, respectively, constitute about 60 and 24% of yolk dry matter (Burley et al., 1993) and about 95 and 5% of yolk cholesterol (Griffin, 1992), with over 90% of the cholesterol present in free form (Bitman and Wood, 1980; Kuksis, 1992). But there were large knowledge gaps. For example, prior to 1986, very little molecular information was available on any of the proteins involved in oocyte growth and systemic lipoprotein transport in the laying hen (Schneider, 1995). It is now known that most of the contents of the oocyte are imported by binding to multifunctional oocyte-specific receptors belonging to the LDL receptor gene family, whereas other proteins (e.g., perlecan and Gallus gallus basement membrane protein 1) are involved in the movement of VLDL particles through the multiple layers of the oocyte prior to reaching the VLDL/VTG receptor on the oocyte surface (Schneider, 2009). Initial experimental approaches attempting to attenuate egg cholesterol levels involved genetic selection studies or feeding hens various nutrients, natural products, nonnutritive factors, or pharmacological agents. Genetic selection for lower egg cholesterol content turned out to not be possible in the short term, but apparently has been achieved to a degree in the long term, as previously discussed with regard to declining cholesterol contents of eggs in the United States. In addition, with the exception of orally administering pharmacological agents to hens, few feeding trials were mechanistically based and therefore did not or could not target key steps in the process by which cholesterol is synthesized, incorporated primarily into VLDL, transported to growing oocytes, and taken up by receptor-mediated endocytosis. As a result, during the past 40 years, the vast majority of experimental approaches to egg cholesterol lowering were ineffective (<10% reduction), as

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reviewed by Shaklee (1972); Naber (1976, 1983); Hargis (1988); Stadelman and Pratt (1989); Griffin (1992); and Elkin (2006, 2007). Exceptions included dietary approaches involving supraoptimal levels of copper (Pesti and Bakalli, 1998), garlic extracts (Chowdhury et al., 2002), or 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (“statin” drugs; Elkin and Rogler, 1990; Elkin et al., 1993, 1999), which resulted in maximal egg cholesterol reductions of 31, 32, and 46%, respectively. However, other researchers have not been able to confirm the copper and garlic work in terms of the magnitude of the reduction in egg cholesterol content attained, whereas residue (radioactivity) was found in egg yolks of hens dosed with [14C]-atorvastatin, the most effective statin in terms of egg cholesterol-lowering ability (Elkin et al., 2003). More recent studies have not fared any better because (1) viable cholesterol-lowering strategies in humans, such as orally administering clofibrate, a peroxisome proliferator-activated receptor-α agonist (König et al., 2007), or feeding phytosterol-enriched diets (Elkin and Lorenz, 2009; Liu et al., 2010), were not efficacious in laying hens, presumably due to physiological differences between species; or (2) various natural products, such as inulin, a fructose polymer (Shang et al., 2010), aqueous alfalfa extract (Deng et al., 2012), barley oil (Walde et al., 2014), high-polyphenol extravirgin olive oil (Laudadio et al., 2015), or annatto (reddish orange food coloring derived from the seeds of Bixa orellana; Hansen et al., 2015), minimally influence hepatic cholesterol biosynthesis or incorporation into VLDL or both, VLDL secretion, or oocytic uptake.

WHAT DOES THE FUTURE HOLD WITH REGARD TO REDUCING EGG CHOLESTEROL? Unfortunately, despite a more complete understanding of the biochemical, cell biological, and molecular genetic aspects of chicken oocyte growth, the availability of the chicken genome sequence, advances in our knowledge of cholesterol trafficking, and new breakthroughs in human cardiovascular therapeutics [e.g., proprotein convertase subtilisin/kexin type 9 (PCSK); Giugliano and Sabatine, 2015], there has been virtually no progress in reducing the cholesterol content of chicken eggs since the author last reviewed this topic (Elkin, 2006, 2007). Most likely, this lack of progress is due to: (1) the potential drawbacks (e.g., residue, cost, and required regulatory approval) of the use of powerful pharmacological agents, such as statins, although the study by Elkin et al. (1999) provided the first proof of principle that egg cholesterol can be lowered by almost 50% with the maintenance of egg production; and (2) the unavailability of chicken strains with “knocked out” or overexpressed key genes involved in cholesterol synthesis and incorporation into VLDL, hepatic VLDL secretion, or oocytic VLDL uptake. Until such avian transgenics become readily available, or a cholesterol-lowering pharmacological agent that is effective, affordable, and residue-free that can be approved for use in poultry, the development and commercial availability of low-cholesterol eggs (i.e., containing >50% less cholesterol) will remain elusive.

PHOSPHATIDYLCHOLINE CONTENT OF EGGS: A NEW CONCERN WITH REGARD TO CARDIOVASCULAR DISEASE PATHOGENESIS? Eggs obviously contain more than cholesterol, and recent work has suggested that phosphatidylcholine, another yolk constituent, may be a participant in a new nutritional pathway contributing to the development of atherosclerosis and incident risks of major adverse cardiovascular events via its conversion by gut microbes to trimethylamine-N-oxide (Wang et al., 2011; Miller et al., 2014). Trimethylamine-N-oxide is toxic to arteries and has been linked to the development of atherosclerosis in the coronary vasculature (Wang et al., 2011; Hazen and Brown, 2014). Thus, manipulation of gut microflora or administering a nonsystemically absorbed inhibitor of the putative pathogenic biochemical pathway represent possible future therapeutic approaches to the prevention of CVD (Wang et al., 2011). In addition, reducing the deposition of phosphatidylcholine into egg yolk represents an alternative strategy for the attenuation of gut trimethylamine-N-oxide formation from consumed eggs. Averaging values from Burley and Vadehra (1989), Watkins (1995), and Kivini et al. (2004), a large egg with a 16.7 g yolk would contain approximately 1 g of phosphatidylcholine. Eggs also are a rich source of choline (147 mg/50 g edible portion; United States Department of Agriculture Agricultural Research Service, 2015), which was recently recognized as an essential nutrient for humans (Iannotti et al., 2014). Although choline can be endogenously synthesized in humans, high requirements during pregnancy, lactation, and in the postnatal period necessitate adequate consumption of this micronutrient (Iannotti et al., 2014). Ironically, because choline is a precursor of phospholipids, including phosphatidylcholine, additional research will be necessary to better understand the relative health risks and benefits of consuming eggs.

ANALYTICAL METHODS Yolk cholesterol: Following lipid extraction, typically with chloroform:methanol (2:1, volume:volume), the cholesterol content of eggs is most commonly determined by spectrophotometric or chromatographic methods. Regardless of the method used, as previously mentioned, researchers should validate their procedure by analyzing a standard reference material such

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as SRM1845a from the National Institute of Standards and Technology. An example of an excellent spectrophotometric method used by the author is that of Rudel and Morris (1973) that employs the reagent, ortho-phthalaldehyde. Although very accurate, a negative characteristic of this assay is that it involves the use of concentrated glacial acetic and sulfuric acids (hint: don’t wear good clothes when performing it). The method also can be used to determine total plasma cholesterol content. An example of an excellent gas chromatographic method is AOAC International official method 994.10 (AOAC International, 2000). Lipid in the sample is saponified in ethanolic potassium hydroxide at high temperature. The unsaponifiable fraction containing cholesterol and other sterols are extracted with toluene, and the sterols are then derivatized to trimethylsilyl ethers and quantified by gas-liquid chromatography. Plasma cholesterol: Cholesterol circulates in the bloodstream as a component of various lipoprotein classes. In assessing risk for CVD in humans, physicians typically order a lipid panel, which includes the determination of total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides in a fasting plasma sample with treatment decisions based primarily on LDL-cholesterol (Warnick and Remaley, 2001). In clinical practice, LDL-cholesterol is often calculated by the Friedewald equation (Friedewald et al., 1972), which requires HDL-cholesterol and triglycerides. However, unlike the “gold standard” reference method for the determination of LDL-cholesterol, the Friedewald procedure does not require ultracentrifugation steps and does not measure the lipoprotein particles directly (Otvos et al., 2002; Oliveira et al., 2013). As a result, because lipoprotein particles are heterogeneous both in size and composition, even the most accurate lipoprotein cholesterol measurements may provide an inaccurate measure of the number of circulating lipoprotein particles and the CVD risk that they confer in an individual (Otvos et al., 2002). Therefore, in order to gain greater insight into individual patient disorders often masked by standard lipid tests, advanced lipoprotein tests are performed and involve methods such as analytic ultracentrifugation, density gradient ultracentrifugation, gradient gel electrophoresis, immunoaffinity chromatography, and two-dimensional gel electrophoresis (Superko, 2009). One approach to the determination of plasma lipoprotein cholesterol and triglyceride distributions in the laying hen was reported by the author in collaboration with scientists from the former Parke-Davis Pharmaceutical Research Division (Elkin et al., 1993). Using the method of Kieft et al. (1991), a high-performance gel filtration chromatography system was used to separate the plasma lipoprotein fractions, whereas the cholesterol and triglyceride contents of the different lipoprotein fractions were determined by enzymatic assays.

CONCLUSIONS l

The response to changes in dietary cholesterol in humans is heterogeneous. In addition to hyperresponders and others at risk of CVD, including diabetics, dietary cholesterol intake should be restricted. l The production of shell eggs with a markedly reduced (i.e., >50%) cholesterol content remains a viable research goal. l Low-cholesterol eggs would be a desirable dietary option for millions of Americans who need to restrict their cholesterol intake but wish to include whole eggs or egg yolks in their diet. l Low-cholesterol eggs would command a premium in the marketplace. l The development and commercial availability of low-cholesterol (>50% reduction) eggs remains elusive due to drawbacks associated with the use of pharmacological agents and the general unavailability of transgenic strains of laying hens. l

ACKNOWLEDGMENT The author wishes to thank Dr. Penny M. Kris-Etherton for her helpful comments on an earlier version of this chapter.

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