1. Introduction

In spite of the removal of the upper limit for cholesterol intake from the dietary guidelines [1], there is still uncertainty regarding egg intake and increased risk for heart disease in individuals with Type 2 Diabetes Mellitus (T2DM) [2,3]. The literature is quite controversial in this topic; some studies report that dietary cholesterol is associated with diabetes risk [4,5] while other studies do not find this connection [6,7]. Although there are reports on clinical interventions which have shown positive effects on the biomarkers of heart disease upon egg consumption in diabetic populations [8,9], there is still a need for more clinical trials to better establish a connection between egg intake and biomarkers of heart disease.

In contrast, oatmeal, a good source of β-glucan, has been characterized as heart healthy due to the effectiveness of this dietary fiber component in lowering plasma LDL cholesterol [10]. Oatmeal has also been shown to be beneficial in the case of uncontrolled T2DM [11]. In our previous crossover study, we demonstrated that compared to an oatmeal-based breakfast, one egg per day did not alter the biomarkers for heart disease or glucose metabolism [9]. Further, egg intake resulted in lower plasma Tumor Necrosis Factor- α (TNF-α) and Aspartate Aminotransferase (AST) values compared to the oatmeal indicating positive effects of eggs on these biomarkers of inflammation [9].

Individuals with diabetes have other conditions that further increase their risk for heart disease including dyslipidemias, oxidative stress and low-grade inflammation [12]. Numerous studies have shown that antioxidants exert a protective effect against oxidative stress and inflammation [13]. Eggs are a very good source of antioxidants including carotenoids, vitamins and minerals. The aim of this study was to investigate further what nutrients and antioxidants present in egg could be responsible for the observed changes in inflammation in diabetic patients when compared to an oatmeal breakfast. For that purpose, we measured plasma carotenoids present in eggs and conducted a careful dietary analysis of other vitamins and minerals present in eggs that could be responsible for the lower concentrations of TNF-α and AST. We hypothesized that plasma lutein and zeaxanthin would be higher following the egg breakfast and that Vitamin A, Vitamin E and selenium would be consumed in higher concentrations during the egg breakfast and therefore contribute to the observed results.

2. Methods

      2.1.  Experimental Design

Thirty-three individuals with type-2 diabetes were recruited to participate in this study. The age was between 35-65 years. We estimated that 25 subjects would be enough to observe changes in inflammatory markers based on previous studies [14]. We recruited 33 subjects to allow for attrition. The study took place between June-December 2013. The exclusion criteria were uncontrolled diabetes, retinopathy, heart disease, cancer, or renal problems. In addition, participants had to have glycosylated Hemoglobin (HbA1c) < 9%. Participants were randomly allocated by one of two breakfasts to consume either one egg or 40 g of oatmeal with 2 cups (472 mL) of lactose-free milk/day for 5 weeks. After a 3-week washout, subjects were allocated to the alternate breakfast for an additional 5 weeks. Subjects were 98% compliant with this intervention. A more detailed description of the experimental design has been reported previously [9].

Twenty-nine subjects finished both arms of this dietary intervention. The intervention protocol was approved by the University of Connecticut Institutional Review Board, the Ethical Committee from Centro de Investigacion en Alimentacion y Desarrollo (CIAD), and the Review Board from Hospital Ignacio Chávez in the city of Hermosillo, Sonora, Mexico. All subjects gave written informed consent prior to initiating the study. This study is registered at Clinicaltrials.gov (trial # NCT02181244)

       2.2.  Dietary Records

Participants were asked to follow their usual diet during both breakfast dietary periods. Four-day weighed food records on alternate days [15] were used to evaluate dietary intake of vitamins and minerals during both breakfast periods for a total of 8 records. Subjects were provided with food scales (Ohaus CS 2000) and a chart to record daily intake and researchers in the study worked very closely with participants to ensure reliable food assessment during the day. Dietary intake was analyzed using the ESHA Food processor program (ESHA, Food Processor, 7.20, ESHA Research Editor, 2007). Regional foods typically consumed by these subjects are also included in this database [16].

       2.3.  Plasma Lutein and Zeaxanthin

Lutein and zeaxanthin were extracted from plasma as described previously [17] using a 2:1 (v:v) chloroform: methanol mixture followed by hexane. A known concentration of internal standard (trans-β-apo-8’-carotenal, Sigma-Aldrich, St. Louis, MO) was added to each sample prior to extraction to calculate carotenoid recovery.

3. Results

      3.1.  Dietary Records

Dietary records indicate substantial differences in vitamin and mineral intake between the two breakfasts. After the egg period there was a 326% increase in vitamin A (p = 0.002), 127% in vitamin E (p < 0.001) and 159% in selenium (p< 0.001). In contrast, calcium concentrations were two-fold and magnesium 1.2-fold higher during the oatmeal period due to the addition of 2 glasses of lactose-free milk (Table 1).

       3.2.  Plasma Antioxidants

Plasma lutein and zeaxanthin (Figure 1A) and dietary lutein + zeaxanthin (Figure 1B) were not different between breakfast periods.

4.  Discussion

       4.1.  Dietary Records

The intention of this short communication was to determine if antioxidants present in eggs could be associated with the lower plasma concentrations of AST and TNF-α compared to oatmeal as found in our previous study [9]. We were able to demonstrate, by use of dietary records, that consumption of vitamin A, vitamin E, and selenium - nutrients present in eggs - was much higher during the egg breakfast. We also have shown that both dietary and plasma lutein and zeaxanthin were not higher in the egg compared to the oatmeal period.

       4.2.  Biomarkers of Inflammation and Lutein and Zeaxanthin

Diabetic patients are exemplified by having elevated concentrations of biomarkers of inflammation [12]. TNF-α contributes to the modulation of immune and inflammatory responses [18] and the primary source of this cytokine is activated macrophages and monocytes [19]. High concentrations of liver enzymes, such as AST, is indicative of liver inflammation [20]. Thus, the observed reductions in plasma concentrations of AST and TNF-α in our previous study following egg [9] deserves further consideration. Originally, we hypothesized that these decreases might be related to the presence of lutein and zeaxanthin in the egg yolk.

Previous studies in our laboratory have shown significant increases in plasma lutein and zeaxanthin after consumption of 2-3 eggs for an extended period of time in children [21], young populations [17], elderly [22], and obese [23] individuals and in subjects classified with metabolic syndrome [24]. Although in this study we did not observe any changes in dietary lutein and zeaxanthin, other studies have reported a consistent increase in plasma lutein and zeaxanthin with egg intake, which is not directly associated to total dietary intake [14,25] but is due to the fact that lutein and zeaxanthin are highly bioavailable in eggs [26]. Although eggs are not an excellent dietary source for lutein and zeaxanthin, the egg matrix possesses the ideal milieu for effective absorption of these carotenoids in the small intestine [24]. However, as previously shown, one egg per day is not enough to result in significant increases of plasma lutein and zeaxanthin. As reported by DiMarco et al. [17] in subjects consuming 0, 1, 2, and 3 eggs for 4 weeks each, significant differences in plasma lutein and zeaxanthin compared to 0 eggs were observed with the intake of 2 and 3 eggs but not with only 1 egg per day. Missimer et al. [27] also reported higher concentrations of plasma lutein and zeaxanthin compared to an oatmeal breakfast after the consumption of 2 eggs per day. It appears that the concentration of lutein and zeaxanthin in one egg is not sufficient to increase plasma concentrations in most cases. Although the concentrations of lutein and zeaxanthin were higher after egg consumption, these numbers did not reach significance suggesting that the observed reductions in inflammatory biomarkers might hot have been related to these carotenoids

       4.3.  Biomarkers of Inflammation and Antioxidants in Eggs

In contrast to lutein and zeaxanthin intake, analysis of dietary records showed that the concentrations of micronutrients varied widely depending on the assigned breakfast. It is clear that those individuals in the egg group consumed higher concentrations of antioxidants including vitamin A, vitamin E, and selenium, which could be associated with previously observed decreases in TNF-α and AST.

Retinoic acid has been demonstrated to attenuate the inflammatory response and reduce the production of inflammatory mediators including TNF-α in various cell types of peripheral tissues [28]. Vitamin A protects against inflammation when provided in reasonable amounts as a supplement or as part of the diet. For example, it has been shown that when great amounts of vitamin A are provided as a supplement, this results in increases in inflammation and liver enzymes in obese women [29] while in cells studies, addition of vitamin A ameliorates cholestatic liver injury and decreases the concentrations of liver enzymes [30]. Vitamin E has been shown to inhibit nuclear factor-ĸβ, reduce the production of TNF-α [31], and reduce concentrations of TNF-α during inflammatory conditions [32]. In addition, a recent meta-analysis also concluded that vitamin E supplementation optimizes liver enzyme levels for those patients identified with non-alcoholic fatty liver disease and hepatic cirrhosis [33] clearly indicating the protective effect of vitamin E against inflammation.

Eggs are an important source of selenium and studies have shown that selenium deficiency exacerbates the inflammatory response [34]. Further, selenium, due to its unique role of incorporation into selenoproteins, has been identified to have multiple health effects [35]. Selenium has also been reported to decrease liver enzymes in diabetic rats [36]. However, it has also been indicated that excess selenium supplementation can lead to insulin resistance and oxidative stress [34], thus obtaining selenium from dietary sources would be a good practice rather than using selenium supplementation.

5.  Conclusions

These studies [27-35] support that the higher concentrations of dietary vitamin A, vitamin E, and selenium during the egg period could be associated with the observed decreases in TNF-α and AST compared to the oatmeal breakfast [9]. In addition, this study further confirms that inclusion of 1 egg/d in people with T2DM increases those nutrients with antioxidant properties, which could be beneficial in mitigating inflammation in this high-at risk population.

Figures 1(A-B): (A) Concentration of plasma lutein and zeaxanthin after 5 weeks of consuming one egg per day or a cup of oatmeal with lactose-free milk and (B) intake of lutein and zeaxanthin during the egg and the oatmeal periods.

Table 1: Vitamin and mineral intake of Type 2 Diabetic subjects (n = 29) during the oatmeal and the egg periods.

1. U. S Department of Health and Human Services; US Department of Agriculture.
2. Shin JY, Xun P, Nakamura Y, He K (2013) Egg consumption in relation to risk of cardiovascular disease and diabetes: A systematic review and meta-analysis. Am J Clin Nutr 98: 146-159.
3. Qureshi AI, Suri MFK, Ajmed S, Nasar A, Divani AA, et al. (2007) Regular egg consumption does not increase the risk of stroke and cardiovascular diseases. Med Sci Monit 131: CR1-CR8.
4. Radzeviciene L, Ostrauskas R (2012) Egg consumption and the risk of type 2 diabetes mellitus: A case-control study. Public Health Nutr 15: 1437-1441.
5. Djousse L, Gaziano JM, Buring JE, Lee IM (2009) Egg consumption and risk of type 2 diabetes in men and women. Diabetes Care 32: 295-300.
6. Djousse L, Kamineni A, Nelson TL, Carnethon M, Mozaffarian D, et al. (2010) Egg consumption and risk of type 2 diabetes in older adults. Am J Clin Nutr 92: 422-427.
7. Zazpe I, Beunza JJ, Bes-Rastrollo M, Basterra-Gortari FJ, Mari-Sanchis A, et al. (2013) SUN Project Investigators. Egg consumption and risk of type 2 diabetes in a Mediterranean cohort: The SUN Project. Nutr Hosp 28: 105-111.
8. Pearce KL, Clifton PM, Noakes M (2011) Egg consumption as part of an energy-restricted high-protein diet improves blood lipid and blood glucose profiles in individuals with type 2 diabetes. Brit J Nutr 105: 584-892.
9. Ballesteros MN, Valenzuela F, Robles AE, Artalejo E, Aguilar D, et al. (2015) One egg per day improves inflammation when compared to an oatmeal-based breakfast without increasing other cardiometabolic risk factors in diabetic patients. Nutrients 7: 3449-3463.
10. Thongoun P, Pavadhgul P, Burmrungpert A, Satitvipawee MP, Harjani Y, et al. (2013) Effect of oat consumption on lipid profiles in hypercholesterolemic adults. J Med Assoc Thai 96: S25-S32.
11. Lammert A, Kratzch J, Selhorst J, Humpert PM, Bierhaus A, et al. (2008) Clinical benefit of a short term dietary oatmeal intervention in patients with type 2 diabetes and severe insulin resistance: A pilot study. Exp. Clin. Endocrinol. Diabetes 116: 132-134.
12. Kontoangelos K, Papageorgiuo CC, Raptis AE, Tsiotra P, Boutati E, et al. (2014) Cytokines, diabetes mellitus and psychopathology: A challenging combination. Neuro Endocrinol Lett 35: 159-169.
13. Siti HN, Kamisah Y, Kamisia J (2015) The role of oxidative stress, antioxidants and vascular inflammation in cardiovascular Disease (a review). Vascul Pharmacol 71: 40-56.
14. Blesso CN, Andersen CJ, Barona J, Volk B, Volek JS, et al. (2013) Effects of carbohydrate restriction and dietary cholesterol provided by eggs on clinical risk factors of metabolic syndrome. J Clin Lipidol 7: 463-471.
15. Bingham SA (1985) The dietary assessment of individuals: methods, accuracy, new techniques and recommendations. Dietary Survey Method 57: 705-741.
16. Hand E (2007) Food Processor II Program, Salem, OR, ESHA Research Editor.
17. DiMarco DM, Norris GH, Millar C, Blesso C, Fernandez ML (2017) 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 147: 323-339.
18. Mancini F, Toro CM, Mabilia M, Giannangeli M, Pinza M, et al. (1999) Inhibition of tumor necrosis factor-α (TNF-α)/TNF-α receptor binding by structural analogues of suramin. Biochem Pharmacol 58: 851-859.
19. Carswell EA, Old LJ, Kasel RL, Green S, Fiore N, et al. (1975) An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci USA 72: 3666-3670.
20. Goessling W, Massaro JM, Vasan RS, D’Agostino RB, Ellison RC, et al. (2008) Aminotransferase levels and 20-year risk of metabolic syndrome, diabetes and cardiovascular disease. Gastroenterology 135: 1335-1944.
21. Ballesteros MN, Cabrera RM, Saucedo MS, Fernandez ML (2004) Dietary cholesterol does not increase biomarkers for chronic disease in a pediatric population at risk from Northern Mexico. Am J Clin Nutr 80: 855-861.
22. Greene CM, Waters D, Clark RM, Contois JH, Fernandez ML (2006) Plasma LDL and HDL characteristics and carotenoid content are positively influenced by egg consumption in an elderly population. Nutr Met 3: 6.
23. Mutungi G, Waters D, Ratliff J, Puglisi MJ, Clark RM, et al. (2010) Eggs distinctly modulate plasma carotenoid and lipoprotein subclasses in adult men following a carbohydrate restricted diet. J Nutr Biochem 21: 261-267.
24. Blesso CN, Andersen CJ, Bolling B, Fernandez ML (2013) Egg intake improves carotenoid status by increasing HDL cholesterol in adults with metabolic syndrome. Food and Function 4: 213-221.
25. Missimer A, Fernandez ML, DiMarco DM, Norris GH, Blesso CN, et al. (2018) Compared to an oatmeal breakfast, two eggs/day increased plasma carotenoids and choline without increasing trimethyl amine-oxide concentrations. J Am Coll Nutr 37: 140-148.
26. Chung HY, Rasmussen HM, Johnson EJ (2004) Lutein bioavailability is higher from lutein-enriched eggs than from supplements or spinach in men. J Nutr 134: 1887-1193.
27. Missimer A, Fernandez ML, DiMarco DM, Norris GH, Blesso CN, et al. (2018) Compared to an oatmeal breakfast, two eggs/day increased plasma carotenoids and choline without increasing trimethyl amine-oxide concentrations. J Am Coll Nutr 37: 140-148.
28. Dheen TS, Jun Y, Yan Z, Tay SSW, Ling EA (2005) Retinoic acid inhibits the expression of TNF-α and iNOS in activated rat microglia. GLIA 50: 21-31.
29. Farhangi MA, Keshavarz SA, Eshraghian M, Ostadrahimi A, Saboor-Yaraghi AA (2013) Vitamin A supplementation, serum lipids, liver enzymes and C-reactive protein concentrations in obese women of reproductive age. Ann Clin Biochem 50: 25-30.
30. Wang G, Xiu P, Li F, Xin C, Li K (2014) Vitamin A supplementation alleviates extrahepatic cholestasis liver injury through Nrf2 activation. Oxidat Med Cell Longev: 273692.
31. Suzuki YJ, Packer L (1993) Inhibition of NF-kappa activation by Vitamin E derivatives. Biochem Biophys Res Commun 193: 277-283.
32. Sarir H, Emdadifard G, Farhangfar H, Taheri-Chadorneshin J (2015) Effect of vitamin E succinate on inflammatory cytokines induced by high intensity interval training. J Res Med Sci 20: 1177-1181.
33. Ji HF, Sun Y, Shen L (2014) Effect of Vitamin E supplementation on aminotransferase levels in patients with NAFLD, NASH and CHC: results from a meta-analysis. Nutrition 30: 986-991.
34. Wei Z, Yao M, Li Y, He X, Yang Z (2014) Dietary selenium deficiency exacerbates lipopolysaccharide-induced inflammatory response in mouse mastitis models. Inflammation 37: 1925-1931.
35. Rayman P (2012) Selenium and human health. Lancet 379: 1256-1268.
36. Zou C, Qiu Q, Chen H, Dou L, Liang J (2016) Hepatoprotective effects of selenium during diabetes in rats. Hum Exp Toxicol 35: 114-123.