The effects of consuming docosahexaenoic acid (DHA)-enriched eggs on serum lipids and fatty acid compositions in statin-treated hypercholesterolemic male patients

Food Research International 38 (2005) 1117–1123 www.elsevier.com/locate/foodres

The eVects of consuming docosahexaenoic acid (DHA)-enriched eggs on serum lipids and fatty acid compositions in statin-treated hypercholesterolemic male patients Leah G. Gillingham a, Linda Caston b, Steve Leeson b, Karen Hourtovenko c, Bruce J. Holub a,¤ a

Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ont., Canada N1G 2W1 b Department of Animal and Poultry Science, University of Guelph, Guelph, Ont., Canada N1G 2W1 c Riverside Cardiac Clinic, Sudbury, Ont., Canada P3E 1H7 Received 7 June 2004; accepted 4 March 2005

Abstract The purpose of this study was to investigate the eVects of consuming a novel docosahexaenoic acid (DHA)-enriched shell egg on the serum lipid levels and
¡ 3 polyunsaturated fatty acids (n ¡ 3 PUFA) of serum phospholipid in statin-treated hypercholesterolemic patients. Fifteen subjects were randomly divided into two treatment groups and consumed either two control or two novel DHA-enriched eggs during two organized breakfast periods of 21 consecutive days each using a double-blinded, cross-over design. The novel enriched eggs from feeding the specialty ration provided 217 mg of DHA and 629 mg of total n ¡ 3 PUFA per day. Total serum cholesterol levels were unchanged with either egg consumption and no signiWcant alterations in lipid levels were found due to a treatment eVect. The novel egg group exhibited a signiWcant rise in eicosapentaenoic acid (EPA) plus DHA levels in serum phospholipid (by 23%) which can be related to a reduced risk for fatal ischemic heart disease. Consumption of this novel egg oVers an alternative food option for more than doubling current sub-optimal DHA intakes in North America.  2005 Elsevier Ltd. All rights reserved. Keywords: Eggs; Docosahexaenoic acid; Humans; Statin; Cholesterol;
¡ 3 polyunsaturated fatty acids; Serum phospholipid

1. Introduction Epidemiological evidence has indicated that increased consumption of Wsh rich in long-chain
¡ 3 polyunsaturated fatty acids (n ¡ 3 PUFA), speciWcally eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), is associated with a reduced risk of cardiovascular disease (CVD) and all-cause mortality (Din, Newby, & Flapan, 2004; Holub, 2002; Lee & Lip, 2003). Lemaitre et al. (2003) reported that higher blood EPA plus DHA levels ¤ Corresponding author. Tel.: +1 519 824 4120x53743; fax: +1 519 763 5902. E-mail address: [email protected] (B.J. Holub).

0963-9969/$ - see front matter  2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.03.006

was associated with a 70% lower risk of fatal ischemic heart disease (IHD) in older adults (Lemaitre et al., 2003). Recently, it has been acknowledged that a laboratory assessment of n ¡ 3 PUFA levels in the blood lipid should be considered as part of a 21st century coronary heart disease risk assessment panel (Harris, 2003). In view of very low intakes of Wsh and DHA (approximately 78 mg/day for US adults) in the population (Raper, Cronin, & Exler, 1992), there is interest in producing n ¡ 3 PUFA eggs via altered feeding (dietary Xaxseed inclusion) as a source of n ¡ 3 PUFA including DHA (Ferrier et al., 1995; Jiang & Sim, 1993; Lewis, Schalch, & Scheideler, 2000). Such eggs can potentially enhance the intake of n ¡ 3 PUFA, particularly DHA,

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for health during pregnancy and lactation (e.g., neuronal beneWts for infants) (Cunnane, Francescutti, Brenna, & Crawford, 2000) as well as potential cardioprotective eVects in the overall population (Din et al., 2004; Holub, 2002; Lee & Lip, 2003). In the present study, hypercholesterolemic patients having their cholesterol controlled via physician-prescribed statin medication were utilized as subjects since such patients are often advised to restrict or cease the consumption of eggs based on the cholesterol content of the yolk. As a result of the rising attention attributed to the cardioprotective potential of the Wsh-based n ¡ 3 PUFAs such as DHA, it was of interest to produce and evaluate, through a clinical trial, the consumption of a novel egg enriched to contain higher levels of DHA (108 mg/egg) since common commercial n ¡ 3 PUFA eggs derived from feeding 10% Xaxseed in the laying hens’ ration typically contain much lesser amounts (Ferrier et al., 1995). The objective of the study herein was to investigate eVects that consumption of the novel DHA-enriched shell eggs versus regular eggs may have on the serum lipid levels and the fatty acid proWles of serum phospholipid (physiological biomarker) in statin-treated hypercholesterolemic male subjects using a double-blind cross-over design.

2. Materials and methods 2.1. Subjects Fifteen male statin-treated hypercholesterolemic patients ranging in age from 35 to 78 were recruited from the Sudbury area by the Riverside Cardiac Clinic (Sudbury, Ontario, Canada). The selected hypercholesterolemic patients were under physician-directed treatment with statin drug therapy for blood cholesterol reduction for more than three months. Patients were advised on no consumption of Wsh products or food concentrated with
¡ 3 fatty acids two weeks prior to the study. One patient withdrew from the study during the second study period due to health problems unrelated to the study. Baseline characteristics of the 14 patients who successfully completed the study included an average age of 61 § 3 years, weight of 104.5 § 6.6 kg and BMI (body mass index) of 34.3 § 2.6 kg/m2 (mean § SEM). Approval of the experimental protocol for the study was obtained from the University of Guelph Research Ethics Committee and each patient gave written informed consent prior to the study. 2.2. Study design A two-period randomized, controlled, double-blind, cross-over study was conducted and displayed in Fig. 1.

Fig. 1. Design of the intervention study.

Each period of the study consisted of the patients providing a 12 h morning fasting blood sample from the antecubital vein on day 0 (initial) and on day 22 (Wnal). In the Wrst period, patients were randomly divided into two treatment groups and consumed either two control eggs or two novel eggs (DHA-enriched
¡ 3 shell eggs) each day during an organized controlled breakfast at 8 a.m. for 21 consecutive days. In the second period of the study, the groups consumed the opposite eggs following the same protocol as period 1. A washout period of 6 weeks divided the two periods, and the patients were instructed to maintain their normal daily diets, statin therapy, and to continue refraining from the consumption of Wsh products and n ¡ 3 PUFA rich foods. Attendance was recorded every morning and if patients were to be absent, the appropriate number of hard-boiled eggs was supplied in advanced or delivered the same morning to ensure compliance. A sensory questionnaire was administered at the end of the study protocol to assess patient acceptance of the novel egg. 2.3. Egg production The treatment eggs were produced by feeding 40 week old Single Comb White Leghorn (SCWL) pullets specialty laying hen diets formulated by the Department of Animal and Poultry Science, University of Guelph. The control diet consisted of a typical corn and soybean diet using ingredients similar to those reported elsewhere (Caston & Leeson, 1990). The n ¡ 3 PUFA enriched diet was similar to that of the control with the addition of

L.G. Gillingham et al. / Food Research International 38 (2005) 1117–1123

Wsh oil (menhaden), ground Xaxseed, and was slightly modiWed to maintain the similar energy balance to that of the control diet. The diets were fed to the SCWL pullets two weeks prior to egg collection. Three replicates of each treatment egg was sampled for fatty acid analysis prior to the study and then repeated at 3–5 week intervals throughout the study for a total of six sampling periods, resulting in 18 samples of both the control and the novel eggs. Lipids were extracted from the treatment eggs and diets using the method of Bligh and Dyer (1959). Fatty acid compositions of the eggs and diets were analyzed using methods based on those previously described (Ferrier et al., 1992). 2.4. Laboratory analysis On the blood draw days (day 0 and day 22) after an overnight 12 h fast, blood was collected by venipuncture into evacuated tubes (Vacutainer; Becton Dickinson, Rutherford, NJ). Samples were then centrifuged at 1330g for 20 min at 25 °C. The recovered serum was divided into aliquots and stored at ¡80 °C until analyzed. Serum total cholesterol, HDL-cholesterol and triglyceride were quantiWed enzymatically with a Hitachi 911 automated discrete computerized analyzer (Roche Diagnostics) at the provincially certiWed Animal Health Laboratory, University of Guelph. LDL-cholesterol was calculated using the Friedewald equation (Friedewald, Levy, & Fredrickson, 1972). Serum lipids were extracted by the method of Bligh and Dyer (1959), lipids isolated by thinlayer chromatography, and the fatty acid compositions of the serum phospholipid (a recognized physiological bio-maker for n ¡ 3 PUFA status) (Arab & Akbar, 2002; Nikkari, Luukkainen, Pietinen, & Puska, 1995) was determined by capillary gas-liquid chromatography after transmethylation as previously described (Young, Maharaj, & Conquer, 2004). 2.5. Statistical analysis Statistical analyses were performed using SAS version 8.2 (SAS Institute, Cary, NC). Baseline comparisons for anthropometric data between the randomized groups were made using an independent t-test. After checking for normality, the triglyceride concentrations were logarithmically transformed before analysis because of skewness. A paired t-test was used to compare initial (day 0) and Wnal (day 22) values for the serum lipids and fatty acid compositions of the serum phospholipid within each treatment group. Analysis of variance (ANOVA) was used to test for a treatment eVect (net change between day 0 and day 22 for the two treatment groups). Egg fatty acid compositions were also analyzed by ANOVA. If there was a signiWcant treatment eVect by F-test, the least-signiWcant diVerence test (Tukey’s test) was used for follow-up comparisons of treatment means.

1119

A carryover eVect was evaluated using baseline values of each period by a paired t-test. The data is shown as group mean § standard error of mean (SEM) and all signiWcant levels are at P 6 0.05 unless otherwise indicated.

3. Results There was no statistical diVerence between the baseline characteristics (age, weight and BMI) of the two randomized treatment groups. Based on the
¡ 3 fatty acid compositions of the serum phospholipids, there was no carryover eVect measured in the study when comparing the baseline (day 0) values between periods 1 and 2, indicating that the washout period was adequate. The fatty acid compositions of the control and novel eggs are shown in Table 1. Two novel eggs provided approximately 217 mg DHA/day, 251 mg EPA + DHA/ day, and 629 mg total n ¡ 3 PUFA/day, whereas the two control eggs provided approximately 61 mg DHA/day, 62 mg EPA+DHA/day, and 100 mg total n ¡ 3 PUFA/ day. Furthermore, the n ¡ 6 to n ¡ 3 PUFA ratio of the novel egg was approximately 2.4:1 versus 14.3:1 for the control egg. The level of the n ¡ 6 PUFA as arachidonic acid (AA) was 60% lower in the novel as compared to the control egg. Serum lipid concentrations for day 0 and day 22 from each treatment group are shown in Table 2. After Table 1 Fatty acid composition of the lipid in the control and the novel eggs (50 g) Fatty acid

Control egg (mg per 50 g egg)

Novel egg (mg per 50 g egg)

16:0 18:0 18:1 18:2 n ¡ 6 (LA) 18:3 n ¡ 3 (
-LNA) 20:3 n ¡ 6 20:4 n ¡ 6 (AA) 20:5 n ¡ 3 (EPA) 22:0 22:4 n ¡ 6 22:5 n ¡ 6 22:5 n ¡ 3 22:6 n ¡ 3 (DHA)  Saturates  Monounsaturates  Polyunsaturates  n ¡ 3 PUFA  n ¡ 6 PUFA Ratio of n ¡ 6 to n ¡ 3 EPA + DHA  Fatty acid

1076.0 § 31.6 351.4 § 10.7 1940.4 § 55.9 622.7 § 20.6 15.6 § 1.8 6.5 § 0.2 84.6 § 2.2 0.6 § 0.1 1.3 § 0.1 5.6 § 0.3 10.1 § 2.0 2.9 § 0.2 30.6 § 1.1 1449.6 § 41.7 2088.2 § 59.7 789.1 § 24.8 50.1 § 2.7 739.0 § 23.1 14.3 § 0.8 31.3 § 1.1 4327.6 § 115.4

1025.7 § 26.3 365.7 § 12.0 1663.7 § 56.9a 641.2 § 22.6 173.9 § 16.9b 5.6 § 0.2a 34.3 § 1.2b 16.9 § 0.5b 1.4 § 0.2 1.7 § 0.1b 0.9 § 0.2b 10.0 § 0.5b 108.4 § 4.2b 1421.2 § 37.7 1816.4 § 59.9a 1005.4 § 38.8b 314.7 § 18.7b 690.8 § 23.9 2.4 § 0.2b 125.3 § 4.6b 4265.6 § 166.3

Values are means § SE (n D 18). LA, linoleic acid;
-LNA,
-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; PUFA, polyunsaturated fatty acid. a,b SigniWcantly diVerent from the control egg, aP < 0.005, bP < 0.0001.

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Table 2 Serum lipid concentrations of statin-treated patients before and after consumption of control or novel eggs Serum lipid

Control (mmol/L)

ModiWed (mmol/L)

Day 0

Day 22

Day 0

Day 22

Total cholesterol LDL-cholesterol HDL-cholesterol Triglyceride Non HDL -cholesterol

4.71 § 0.26 2.64 § 0.17 1.07 § 0.12 0.59 § 0.17 3.64 § 0.29

4.67 § 0.22 2.59 § 0.14 1.11 § 0.13 0.63 § 0.15 3.56 § 0.22

4.62 § 0.26 2.54 § 0.18 1.10 § 0.11 0.62 § 0.16 3.52 § 0.27

4.76 § 0.22 2.76 § 0.17 1.15 § 0.12¤ 0.52 § 0.13 3.61 § 0.22

Values are means § SE (n D 14). LDL, low-density lipoprotein; HDL, high-density lipoprotein. No signiWcant diVerences due to a treatment eVect, P < 0.05. ¤ SigniWcantly diVerent from day 0 within each treatment group, P < 0.05.

consumption of two novel eggs/day for 21 consecutive days, there was a signiWcant increase in serum HDL-cholesterol concentrations by 4.5% (P < 0.05). Furthermore, there was a trend towards a decrease in serum triglyceride levels by approximately 14% and an increase in serum LDL-cholesterol levels by approximately 9% after the period of novel egg consumption; however, these measures were not statistically signiWcant. There was no signiWcant change in serum lipid concentrations after consumption of the control egg. As well, there was no signiWcant change in serum lipid concentrations due to a treatment eVect (i.e., diVerence between consuming the control and novel egg).

Fatty acid compositions of the serum phospholipid are displayed in Table 3. The only fatty acid parameters to exhibit both a signiWcant treatment and a time eVect (net change from day 0 to day 22) are exhibited in Fig. 2. After consumption of two novel eggs/day for 21 consecutive days, there was a signiWcant increase in the total n ¡ 3 PUFA levels by 17% (4.72 § 0.22 to 5.54 § 0.31% by weight, P < 0.005), a signiWcant decrease in the ratio of n ¡ 6 to n ¡ 3 PUFA levels by 15% (7.69 § 0.37 to 6.57 § 0.41% by weight, P < 0.005), a signiWcant increase in DHA levels by 22% (2.71 § 0.13 to 3.31 § 0.22 % by weight, P < 0.01), and a signiWcant increase in the EPA+DHA levels by 23% (3.50 § 0.18 to 4.30 § 0.30 % by weight, P < 0.005). The patients reported no adverse aVects from consumption of either treatment eggs and after the administered sensory questionnaire, the majority failed to correctly identify in which period they had been given the novel DHA-enriched egg. Furthermore, the blinded participants awarded a mean acceptability score of 4.25 (out of 5 as desirable) for the control egg and 4.17 for the novel egg.

4. Discussion The novel DHA-enriched egg produced and tested in our current study (see Table 1) provided 108 mg of DHA which was substantially higher than that of the control

Table 3 Fatty acid composition of the serum phospholipid from statin-treated patients before and after consumption of the control and novel eggs Fatty acid

16:0 18:0 18:1 18:2 n ¡ 6 (LA) 18:3 n ¡ 3 (
-LNA) 20:3 n ¡ 6 20:4 n ¡ 6 (AA) 20:5 n ¡ 3 (EPA) 22:0 22:4 n ¡ 6 22:5 n ¡ 6 22:5 n ¡ 3 22:6 n ¡ 3 (DHA)  Saturates  Monounsaturates  Polyunsaturates  n ¡ 3 PUFA  n ¡ 6 PUFA Ratio of n ¡ 6 to n ¡ 3 EPA + DHA

Control (% by weight of total fatty acids)

Novel (% by weight of total fatty acids)

Day 0

Day 22

Day 0

Day 22

27.84 § 0.34 13.89 § 0.24 12.37 § 0.23 17.69 § 0.53 0.19 § 0.16 3.08 § 0.20 12.39 § 0.39 1.13 § 0.17 1.19 § 0.06 0.33 § 0.03 0.62 § 0.04 0.95 § 0.04 3.36 § 0.24 45.06 § 0.26 14.72 § 0.26 40.22 § 0.26 5.75 § 0.43 34.47 § 0.54 6.40 § 0.43 4.49 § 0.40

27.50 § 0.53 13.90 § 0.32 12.20 § 0.18 17.94 § 0.54 0.18 § 0.02 3.23 § 0.18 12.83 § 0.43 0.86 § 0.08 1.19 § 0.06 0.34 § 0.02 0.63 § 0.04 0.86 § 0.04c 3.54 § 0.22 44.65 § 0.30 14.43 § 0.21 40.92 § 0.32a 5.57 § 0.31 35.36 § 0.40 6.59 § 0.34 4.40 § 0.28

27.85 § 0.38 13.70 § 0.25 12.42 § 0.35 17.85 § 0.57 0.16 § 0.02 3.30 § 0.17 12.60 § 0.40 0.79 § 0.06 1.39 § 0.07 0.39 § 0.02 0.73 § 0.02 0.93 § 0.04 2.71 § 0.13 45.15 § 0.22 14.85 § 0.33 40.00 § 0.37 4.72 § 0.22 35.28 § 0.46 7.69 § 0.37 3.50 § 0.18

27.76 § 0.37 14.03 § 0.27a 12.05 § 0.20 17.23 § 0.45 0.16 § 0.01 3.35 § 0.19 12.64 § 0.41 0.99 § 0.12d 1.29 § 0.05a 0.37 § 0.02 0.73 § 0.03 0.93 § 0.04d 3.31 § 0.22bd 45.31 § 0.26 14.41 § 0.21 40.28 § 0.29 5.54 § 0.31ce 34.75 § 0.30d 6.57 § 0.41cf 4.30 § 0.30ce

Values are means § SE (n D 14). LA, linoleic acid;
-LNA,
-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; PUFA, polyunsaturated fatty acid. No carryover eVect was measured between baseline values from period 1 to period 2. a–c SigniWcantly diVerent from day 0 within each treatment group, aP 6 0.05, bP < 0.01, cP 6 0.005. d–f SigniWcantly diVerent due to a treatment eVect, dP 6 0.05, eP 6 0.01, fP < 0.005.

L.G. Gillingham et al. / Food Research International 38 (2005) 1117–1123

Total n-3 PUFA

*

120 110 100 90 80

Ratio of n-6 to n-3 PUFA 130

Relative Percent

Relative Percent

130

70

120 110 100

*

90 80 70

60

60 Day 0

Day 22-Control

Day 22-Novel

Day 0

Day 22-Control

DHA

*

120 110 100 90 80 70

Relative Percent

140

130

Day 22-Novel

EPA + DHA

140

Relative Percent

1121

*

130 120 110 100 90 80 70

60

60 Day 0

Day 22-Control

Day 22-Novel

Day 0

Day 22-Control

Day 22-Novel

Fig. 2. Relative percent change in fatty acid levels of serum phospholipid from statin-treated patients after consumption of the control and novel eggs. Error bars are standard errors (n D 14). * SigniWcantly diVerent from day 0 and from the net change (day 0–day 22) after control egg consumption.

(31 mg) and that from laying hens fed 10% Xaxseed (81 mg) (Ferrier et al., 1995). Interestingly, while the novel egg was higher in EPA (17 mg) as compared to the control (1 mg) and 10% Xaxseed (10 mg) (Ferrier et al., 1995), it is noteworthy that the EPA:DHA ratio was only 1:6 in the egg yet 1:1 in the feed. This discrepancy may possibly reXect selective transport mechanisms favouring DHA over EPA for embryonic and neuronal development. Since the feeding of high levels of alpha-linolenic acid (
-LNA) from Xax oil did not increase DHA levels in plasma phospholipid (Chan et al., 1993), the signiWcant increase in DHA levels in serum phospholipid seen here most likely arises almost exclusively from the DHA and not the
-LNA in the novel egg enriched in n ¡ 3 PUFA. Furthermore, the very low levels of
-LNA in serum phospholipid was unaltered after consumption of the novel eggs providing 348 mg
-LNA/day versus 31 mg
-LNA /day via the control eggs. None of the measured risk factor for coronary heart disease (Austin, Hokanson, & Edwards, 1998; Executive summary (NCEP), 2001; Fager & Wiklund, 1997; Frost & Havel, 1998; Viles-Gonzalez, Fuster, Corti, & Badimon, 2003) exhibited signiWcant changes due to a treatment eVect (see Table 2). Lewis et al. (2000) observed a 7% increase in serum LDL-cholesterol and a 15% decrease in serum triglyceride levels from 25 hypercholesterolemic subjects not on statin therapy after consumption of 12 n ¡ 3 PUFA enriched eggs per week from

Xaxseed fed hens for 6 weeks. In previous studies from our group, the consumption of four LNA-enriched eggs by normolipidemic subjects per day resulted in no statistically-signiWcant alteration in mean plasma cholesterol or triglyceride levels (Ferrier et al., 1995). On the other hand, consumption of four DHA-enriched eggs per day was associated with a signiWcant decrease in plasma triglyceride concentrations (Oh, Ryue, Hsieh, & Bell, 1991). However, the DHA-enriched eggs in the latter study were produced by inclusion of 10% Wsh oil (MaxEPA) which would most likely be associated with the egg possessing a severe oV-Xavour (Wshy taste) beyond consumer acceptability. Inclusion of 6% Wsh oil into the laying hens’ diet has yielded an oV-Xavour in the enriched egg (Adams, Pratt, Lin, & Stadelman, 1989) and panelists taking part in a sensory evaluation could mildly diVerentiate n ¡ 3 PUFA enriched eggs from dietary addition of 3% menhaden oil (Van Elswyk, Sams, & Hargis, 1992). In the present study, the level achieved in the novel egg did not produce an oV-Xavour that was distinguishable by the patients after a sensory questionnaire. A number of epidemiological studies have reported a strong inverse relation between blood lipid long-chain n ¡ 3 PUFA (Lemaitre et al., 2003; Rissanen, Voutilainen, Nyyssonen, Lakka, & Salonen, 2000; Simon et al., 1995) including EPA plus DHA and heart disease risk. Very recently, Lemaitre et al. (2003) reported that higher levels (4.6% by weight) of combined EPA plus DHA in

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L.G. Gillingham et al. / Food Research International 38 (2005) 1117–1123

plasma phospholipid (physiological biomarker) was associated with a 70% lower risk of fatal IHD relative to a summed value of 3.55% by weight. Therefore, assuming linearity between these two extremes, it may be estimated from the present shiftings (see Table 3) in the EPA plus DHA sums in serum phospholipid (from 3.50 to 4.30%) following consumption of the novel egg that the risk for fatal IHD may be decreased by approximately 53%. However, there are obvious limitations in extrapolating the Wndings from a 3 week trial to chronic levels of EPA+DHA maintained in the circulation over many years and their relationship to IHD risk. In addition to the cardioprotective beneWts attributed to n ¡ 3 PUFA, DHA is highly concentrated in the brain and retina and is physiologically essential for neuronal functioning and visual acuity, respectively (Cunnane et al., 2000; Horrocks & Farooqui, 2003; Salem, Litman, Kim, & Gawrisch, 2001). Due to the importance of DHA for central nervous tissue development in the fetus, the DHA status of the infant is dependent on maternal DHA intake during pregnancy and lactation. Therefore, the international society for the study of fatty acids and lipids (ISSFAL) advised an adequate intake (AI) for DHA of 300 mg/day for pregnant and lactating women (Simopoulos, Leaf, & Salem, 1999), whereas the Institute of Medicine suggested an AI of 140 and 130 mg/day of combined DHA and EPA for pregnant and lactating women, respectively (National Academy of Sciences, 2003). It has been estimated that the per capita intake of DHA is only 48 mg/day for pregnant women (Lewis, Widga, Buck, & Frederick, 1995) and 78 mg/day for US adults (Raper et al., 1992). Consumption of two of the novel DHA-enriched eggs as reported herein could increase current daily DHA intakes by 450% and 277%, respectively, for the aforementioned two groups. In conclusion, consumption of two of the novel DHAenriched eggs was found to signiWcantly increase the overall physiologically-measured
¡ 3 PUFA status, speciWcally DHA, without signiWcantly altering circulating cholesterol levels in a small number of statin-treated hypercholesterolemic patients. Consumption of the novel DHA-enriched eggs can greatly enhance current dietary DHA intakes from non-Wsh sources and help approach or surpass recommended intakes for optimal human health.

Acknowledgements We thank Lise Thompson, Mika Koskela, and Kari Koskela for the organization of the breakfast periods, our subjects for their time and commitment to the entirety of this study, Burnbrae Farms Ltd. (Mississauga, Ontario, Canada) for providing a research grant to support the study, the Advanced Foods Materials Network, and Lipid Analytical Laboratories of the University of Guelph Research Park, University of Guelph.

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