Changes in serum lipids and platelet fatty acid composition following consumption of eggs enriched in alpha-linolenic acid (LnA)

Food Research International 25 (1992) 263-268

Changes in serum lipids and platelet fatty acid composition following consumption of eggs enriched in alpha-linolenic acid (LnA) Les K. Ferriera, Linda Castona, Steve pesona, E. James fquiresa, Bernadette Celib, Lisa Thomas & Bruce J. Holub ‘Department of Animal and Poultry Science, bDepartment of Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada NIG 2 WI

Dietary n-3 fatty acids as found in fish oils have been associated with a reduced risk of coronary heart disease. In this study, serum and platelet lipid profiles were monitored in subjects consuming eggs enriched in alpha-linolenic acid (LnA, 18 : 3 n-3) and, to a more limited extent, other n-3 fatty acids. Eggs were produced by the inclusion of ground flax seed (containing LnA) in the laying hens’ diet. Consumption of LnA-enriched (modified) eggs, but not regular eggs, for 1 or 2 weeks resulted in a marked decline (by 35%) in serum triglyceride levels (significant, p < 0.005, after 1 week) with no change in total or high-density lipoprotein (HDL) cholesterol. A significant rise, p I 0.01, (by about 60%) in the n-3 fatty acid, docosahexaenoic acid (DHA, 22 : 6 n-3), of platelet phospholipid also occurred in subjects consuming the modified eggs. Since serum triglyceride level is becoming recognized as a predictor of coronary heart disease, while DHA is physiologically essential in the brain plus retina as well as a contributor to the dampening of platelet reactivity, these findings may have potential health implications. Keywords : alpha-linolenic

acid, docosahexaenoic

acid, plasma triglyceride, platelet phospholipid acid.

INTRODUCTION

fatty

fatty acids, eicosapentaenoic acid (EPA, 20: 5 n-3) and docosahexaenoic acid (DHA, 22: 6 n-3), have been shown to consistently reduce serum triglyceride levels (Sanders & Roshanai, 1983; Holub et al., 1987) and increase platelet n-3 fatty acid content (Harris, 1989). While there appears to be inconsistent effects of dietary n-3 fatty acids on serum total and low-density lipoprotein (LDL) cholesterol, a mild high-density lipoprotein (HDL)elevating effect has been observed (Weaver & Holub, 1988). These serum and platelet lipid alterations have been associated with a reduced risk of myocardial infarction and thrombosis (Bang et al., 1980; Aberg et al., 1985). In addition, DHA is physiologically essential in the membrane phospholipid of the brain and retina for mental performance and visual acuity, respectively (Health and Welfare Canada, 1990). Enrichment of eggs with EPA and DHA has been accomplished through the inclusion of fish oil containing these fatty acids in laying hens’

Concern over the relationship between dietary cholesterol and coronary heart disease has contributed to the marked decline in consumption of shell eggs over the past decade (Hargis & Van Elswyk, 1991). Although attempts to reduce cholesterol content of egg yolk have been relatively unsuccessful (Hargis, 1988), manipulation of the n-3 fatty acid content of eggs has shown promise in improving their nutritional quality (Hargis & Van Elswyk, 1991). Increased dietary n-3 fatty acid consumption has been shown to offer potential in reducing the risk of coronary heart disease (Dyerberg & Bang, 1979; Bang et al., 1980; Herold & Kinsella, 1989; Kinsella et al., 1990) even in the presence of high levels of cholesterol in the diet (Nestel, 1986). The consumption of the n-3 Food Research International 0963-9969/92/%05.00 0 1992 Canadian Institute of Food Science and Technology 263

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L. K. Ferrier, L. Caston, S. Leeson, E. J. Squires, B. Celi, L. Thomas, B. J. Holub

diets. Consumption of these EPA- and DHA-enriched eggs by humans was recently shown to moderately lower plasma triglyceride levels (Oh et al., 1991); the effect on blood platelets was not studied. However, the detection of an off-flavour in such eggs by the consumer may render them less desirable than regular eggs (Van Elswyk et al., 1990). The ingestion of the n-3 fatty acid, alpha-linolenic acid (LnA, 18 : 3 n-3) may also reduce the risk of coronary heart disease through its conversion to the long chain polyenoic fatty acids, EPA and DHA (Tinoco, 1982; Sanders & Roshanai, 1983; Weaver et al., 1990). The inclusion of ground flax seed (high in LnA) in the laying hens’ diet was shown to produce eggs very high in LnA. Eggs from hens fed a 20% flax seed diet had a linoleic acid (LA, 18 : 2 n-6)/LnA ratio of 1.7/l versus 37/l for control eggs (Caston & Leeson, 1990). Although less pronounced than for LnA, significant increases in DHA levels were also observed in these modified eggs. In this study, it was of interest to study the effect of the consumption of LnA-enriched eggs on serum and platelet lipid profiles in human subjects.

Weeks 1, 2, 5 and 6 of the study were designated dry-out periods (no experimental egg consumption) to allow subjects’ serum and platelet lipids to adapt to the restricted diet described above. During weeks 3, 4, 7 and 8, subjects consumed the experimental eggs. During weeks 3 and 4, control eggs were consumed at 4/day (providing 0.04 g LnA/day), while 4 modified eggs/day (providing 1.1 g LnA/day) were consumed during weeks 7 and 8. The corresponding DHA intakes for 4 eggs were 27 mg (control) and 94 mg (modified), respectively. A variety of cooking methods were employed including baking, boiling and frying since it appears that variable cooking methods do not alter the fatty acid composition or functional properties of eggs (Hargis & Van Elswyk, 199 1). Control and modified eggs were produced according to the method of Caston & Leeson (1990); hens producing control eggs were fed a standard layer diet while hens producing modified eggs were fed a diet containing 20% (w/w) ground flax seed. Egg lipid analysis

MATERIALS

AND METHODS

Materials

Siliconized vacutainer tubes were purchased from Becton-Dickinson and Co., Rutherford, NJ. HDLCholesterol Reagent and Cholesterol High Performance CHOD-PAP Kit were from Boehringer Mannheim, Dorval, Quebec. Merck silica gel 60 TLC plates were obtained from BDH Chemicals, Toronto, Ontario. Standard fatty acid methyl esters were from NuChek Prep Inc., Elysian, MN. All chemicals and solvents were of analytical grade. Subjects and experimental design

The subjects in this study were five healthy male volunteers with an average age of 45.8 f 3.0 years and an average weight of 80.4 f 3.0 kg (values are mean f SEM). The subjects were instructed to refrain from consuming eggs (other than the experimental eggs), canola oil, fish, alcohol, aspirin and all other medications throughout the experimental period of 8 weeks and to refrain from rigorous physical training. All other dietary habits were maintained at a constant level. None of the subjects smoked cigarettes.

Lipids were extracted from eggs by the method of Bligh & Dyer (1959). The cholesterol concentration of eggs was determined by high performance liquid chromatography according to the method of Chung et al. (1991). Egg lipids were transmethylated in the presence of 6% (by volume) H,SO, in methanol and a known amount of the internal standard monopentadecanoin for 12 h at 80°C. Fatty acid methyl esters derived from the phospholipid fraction were analyzed isothermally on a Hewlett-Packard 5890 gas liquid chromatograph (Hewlett-Packard Co., Palo Alto, CA) equipped with a megabore column (Chromatographic Specialties Inc., Brockville, Ontario) and identified by comparison of their retention times with those of known standards. Serum lipid analysis

Fasting blood samples were drawn from the antecubital vein of human subjects on day 0 and at the end of each subsequent week of the experimental period into siliconized vacutainer tubes. Blood was incubated at room temperature (20-24°C) for 5 h to allow clotting to occur and then centrifuged at 350 g to obtain serum. Serum triglyceride and total cholesterol concentrations were quantitated on a DACOSR (Coulter Electronics, Burlington,

Lipid changes with linoleate-enriched egg consumption

Ontario), chemistry analyzer using modifications of the Fossati & Prencipe (1982) and McGowan et al. (1983) reagent methods. HDL cholesterol was isolated by precipitation of all other lipoproteins from serum using an HDL-Cholesterol Reagent and quantitated enzymatically using a Cholesterol High Performance CHOD-PAP Kit. Platelet fatty acid analysis Fasting blood samples were drawn, as above, on Day 0 and at the end of each week of the experimental period into siliconized vacutainer tubes containing l/lOth the volume of 38% (w/v) trisodium citrate as anticoagulant. Whole blood was centrifuged at 250 g for 7 min at room temperature (2&24”(Z) to obtain platelet-rich plasma (PRP), to which Na,EDTA was added at a final concentration of 6 mM. After refrigeration at 4°C for 1 h, the PRP was centrifuged at 2000 g for 15 min at 4°C. The platelet pellet obtained was resuspended in a Tris/ saline buffer (0.15 M NaCl, 0.02 M Tris/HCl, 2 mM Na,EDTA, pH 7.4) and centrifuged at 2000 g for 15 min at 4°C. The final platelet pellet was resuspended in a glucose-containing buffer (123 ITIM NaCl, 15 mM Tris/HCl, 5 mM glucose, pH 7.4) prior to extraction of the platelet lipids by the method of Bligh & Dyer (1959). The lipid extracts were applied to Merck pre-coated silica gel 60 plates and separated into their various lipid components using hep tane/isopropyl ether/acetic acid (60 : 40 : 3, v/v/v) as the developing mixture. The lipid classes were detected under ultraviolet light after spraying with 2’,7’-dichlorofluorescein and exposing to ammonia vapour. The total phospholipid band was then scraped from the plate, transmethylated for 3 h at 80°C and the derived fatty acid methyl esters analyzed by gas liquid chromatography as described above. Statistical analyses All serum lipid and platelet fatty acid data were analyzed by analysis of variance and statistical differences were assessed by a paired Student’s t-test.

RESULTS The fatty acid compositions of control modified egg yolk are shown in Table 1. The of LnA in modified eggs was 27-fold higher that of control eggs being 8.2 and 0.3 weight

and level than % of

265

Table 1. Levels of selected fatty acids in control and modified egg yak” Fatty acid

Weight % of total fatty acids Control

18: ln-9 18 : 2n-6 18 : 3n-3 20 : 4n-6 20 : 3n-3 20 : k-3 22 : 5n-6 22 : 6n-3 Total polyunsaturates

35.4 9.5 0.3 0.9

* + f * tr tr 1.2 f 0.2 f. 12.1 *

0.9 0.6 0.0 0.1

0.1 0.1 0.4

Modified 32.4 + 0.9 11.7 rt 0.3** 8.2 + 0.6** 0.5 z!Io.o** tr tr 0.7 f 0.1* 0.7 + 0.1** 22.0 I!z1.0**

“Eggs were from laying hens fed a standard layer diet (control eggs) or a 20% flax seed diet (modified eggs). The data represent the mean f SEM of determinations from five separate eggs. *Significantly different from control (p 5 0.05). **Significantly different from control @ 5 0.01). tr = trace (< 0.2 weight %).

total fatty acids, respectively. The LA/LnA ratio of control and modified eggs was 32/l and 1.4/l, respectively, and is in general agreement with our earlier data (Caston & Leeson, 1990). The level of DHA was also significantly (p 5 0.01) elevated (by 3.6-fold) in the modified eggs. Eggs from hens fed the flax seed diet had significant @II 0.01) decreases in the n-6 fatty acids, 20: 4 n-6 and 22 : 5 n-6, as compared to control eggs. Overall, the n-6/n-3 fatty acid ratio for modified eggs (1.4/l) was much less than that of control eggs (23/l). The total polyunsaturate level was elevated (by 10 weight Oh) in modified eggs versus control eggs, due mainly to an increase in LnA (8 weight %) and LA (2 weight %). There was no significant (p > 0.05) differences in total egg yolk weight (15.9 f 0.6 g versus 15.9 + 0.1 g) or the % fat (37.7 + 1.3% versus 36.9 f 0.9%) and cholesterol level (182 + 37 mg versus 212 f 14 mg) in the yolks of the control and modified eggs, respectively (all values are mean & SEM). Table 2 shows the serum lipid concentrations of subjects before (Day 0) and after 1 week (Day 7) and 2 weeks (Day 14) of egg consumption. Consumption of modified eggs was markedly lowered (by 35%) serum triglyceride levels (significant (p I 0.05) decline after 1 week), with no further decrease observed in the second week compared to the first. No significant @ > 0.05) change in serum triglyceride was evident following the ingestion of control eggs. Total and HDL cholesterol levels rose by 0.3 mmol/litre after 2 weeks of control egg

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L. K. Ferrier, L. Caston, S. Leeson, E. J. Squires, B. Celi, L. Thomas, B. J. Holub

Table 2. Serum lipid concentrations of subjects following consumption of regular or LuA-enriched eggs“ Day 7

Day 14

Triglyceride (mmol/litre) 1.7 f 0.3 Control 2.0 + 0.3 Modified

1.5 z!Y 0.2 1.3 f 0.2*

1.5 + 0.3 1.3 f O.lb

Total cholesterol (mmoVlitre) 6.0 f 0.5 Control 6.2 + 0.3 Modified

6.1 + 0.4 6.6 * 0.4

6.3 + 0.3* 6.4 f 0.2

HDL cholesterol (mmoVlitre) 1.5 * 0.2 Control 1.5 + 0.2 Modified

1.6 f 0.2 1.6 f 0.2

1.8 + 0.3* 1.7 * 0.3

Day 0

Table 3. Fatty acid composition of total platelet pbospholipids following consumption of regular or LuA-enriched eggs” Mol % of total fatty acids

Fatty acid

Day 0 18 : ln-9 18 : 2n-6 18 : 3n-3 20 : 4n-6 20 : 5n-3

“Subjects consumed control or LnA-enriched (modified) eggs for 2 weeks. The data represent the mean f SEM of determinations from five separate subjects. “p 5 0.05 compared to day 0. *Significantly different from day 0 @ I 0.05).

22 : 4n-6 22 : 5n-3 22 : 6n-3

consumption. There were no significant (p > 0.05) changes in total cholesterol or HDL cholesterol levels after consumption of the modified eggs. The fatty acid composition of platelet phospholipids before (Day 0) and after 1 week (Day 7) and 2 weeks (Day 14) of egg consumption are presented in Table 3. The platelet LnA content was very low (0.1 mol% of total fatty acids) prior to egg consumption (Day 0) and remained unchanged following either the ingestion of control or modified eggs for up to 2 weeks. Similarly, the level of n-6 fatty acids were not altered when either eggs were consumed, nor was the level of the n-3 fatty acid, EPA. However, a very significant (p I 0.01) rise in DHA (approximately 60% higher than Day 0) was apparent following the ingestion of modified eggs for 1 or 2 weeks, with no further increase observed in the second week compared to the first. In contrast, there was no change in platelet DHA following control egg consumption. The level of 22: 5 n-3 also rose significantly (p I 0.05) after 1 week of modified egg consumption, but was not significantly (p > 0.05) elevated following 2 weeks of consumption.

DISCUSSION

In this study, we investigated the effect of consuming LnA-enriched versus conventional eggs on serum lipid profiles and platelet (phospholipid) fatty acid composition. Flax seed is an abundant source of LnA (55% of total fatty acids). Enrichment of hens’ eggs with LnA was accomplished through the inclusion of

Control Modified Control Modified Control Modified Control Modified Control Modified Control Modified Control Modified Control Modified

14.4 + 14.6 f 4.4 + 4.2 f 0.1 * 0.1 * 16.5 f 15.0 + 0.2 f 0.2 + 1.8 f 1.8 f 0.9 f 0.9 + 0.9 + 0.8 *

0.8 0.5 0.4 0.4 0.0 0.0 1.3 0.7 0.0 0.0 0.2 0.1 0.1 0.0 0.1 0.0

Day 7 14.3 + 0.9 14.2 z!z0.5 3.9 f 0.1 4.4 + 0.2 0.1 z!z0.0 0.1 z!I0.0 15.8 f 1.5 16.9 + 0.8 0.1 * 0.0 0.3 * 0.1 1.8 f 0.2 1.8 f 0.1 0.9 * 0.2 1.1 f 0.1* 0.9 f 0.1 1.3 + 0.1**

Day 14 14.3 + 0.5 14.3 f 0.4 4.1 f 0.2 4.5 * 0.1 0.1 * 0.0 0.1 f 0.0 15.1 f 0.3 16.2 f 0.9 0.2 + 0.1 0.3 f 0.0 1.7 * 0.1 1.8 + 0.2 0.7 I!I0.1 1.0 f 0.1 0.8 + 0.1 1.3 f 0.1**

‘Subjects consumed control or LnA-enriched (modified) eggs for 2 weeks. The data represent the mean + SEM of determinations from five separate subjects. *Significantly different from day 0 (p I 0.05) **Significantly different from day 0 (p I 0.01).

ground flax seed in the laying hens’ diets (Caston & Leeson, 1990). In addition, an increased level of the long chain n-3 fatty acid, DHA, was observed in the eggs (see Table 1) as reported previously (Caston & Leeson, 1990). This was probably due to the desaturation and elongation of LnA to DHA within the hen (Tinoco, 1982) since flax seed is devoid of long chain n-3 fatty acids. The fatty acid alterations in modified eggs resulted in a substantial decline in the n-6/n-3 ratio in egg yolk from 23/l to 1.4/l. Long chain n-3 fatty acids, particularly DHA, appear to be physiologically essential for brain function and visual acuity. Recent nutritional recommendations released by Health and Welfare Canada (1990) recognize the essentiality of n-3 fatty acids in human diets and suggest a dietary n-6/n-3 ratio ranging between 4/l and 10/l. Currently, the ratio is approximately 12/l in the Canadian diet due mainly to the higher n-6 fatty acid (mostly as LA) content relative to n-3 fatty acid (mostly as LnA) in the individual foods we consume. In the light of these recommendations, the substantial reduction in the n-6/n-3 fatty acid ratio of egg yolk from hens fed a flax seed diet greatly improves the nutritional quality of these eggs in this regard compared to regular eggs. Based on the Canadian nutritional recommendations, it can be calculated that the regular and modified eggs (4 eggs/day) provided approximately

Lipid changes with linoleate-enriched egg consumption

5 and 90%, respectively, of the daily recommended n-3 fatty acid intake for adults (Health and Welfare Canada, 1990). Consumption of LnA-enriched eggs by our subjects resulted in a signillcant (p I 0.05) reduction in serum triglyceride levels. It is now becoming recognized that the serum triglyceride level is a significant predictor of coronary heart disease (Austin, 1991). Decreased triglyceride in serum has been associated with a reduced risk of cardiovascular disease, suggesting that the ingestion of LnA-enriched eggs may possibly offer some protection against this risk factor for heart disease. Ingestion of control eggs, in contrast to modified eggs, moderately raised both total and HDL cholesterol levels (see Table 2). The small (5%) rise in serum total cholesterol in control egg consumers is in agreement with the data of Van Elswyk et al. (1990). Increases in total cholesterol and HDL-cholesterol levels have been positively and inversely correlated, respectively, to the risk of heart disease (Gordon et al., 1977; Kannel et al., 1979). The substantial reduction (by 35%) in serum triglyceride concentration (Table 2) in the consumers of the modified eggs occurred after 1 or 2 weeks of egg ingestion (providing 1.1 g LnA/day). The ingestion of linseed oil (providing 9.4 g LnA/day) for 2 weeks failed to change the level of plasma triglyceride in subjects in one study (Sanders & Roshanai, 1983) but, at higher levels in another study, did show a reduction (Singer et al., 1986). Linseed oil is devoid of long chain fatty acids, including DHA (Anderson et al., 1989) while hens fed the flax seed diet produced eggs which were also enriched in DHA compared to control eggs. The presence of increased DHA and LnA in the modified eggs, plus other unknown factors, could have both contributed to the reduction in serum triglyceride observed. In this respect, significant decreases in serum triglyceride have occurred following the ingestion of a fish oil concentrate containing EPA and DHA (Sanders & Roshanai, 1983; Holub et al., 1987). Similarly, the consumption of EPA and DHA enriched eggs has been documented to moderately reduce triglyceride levels in the blood (Van Elswyk et al., 1990; Oh et al., 1991). Activation of blood platelets and aggregation is vital for the normal haemostatic mechanisms involved in blood clotting. However, excessive platelet aggregation can lead to arterial thrombosis (Fuster & Chesebro, 1981). Alterations in platelet phospholipid fatty acid composition ap-

267

pear to affect platelet aggregation. Enrichment of platelet phospholipid with EPA and DHA, via consumption of a fish oil concentrate, was shown to result in a reduction in platelet aggregation (Skeaff & Holub, 1988). Consumption of DHA in viva or its addition to platelets in vitro dampens platelet reactivity (Gaudette & Holub, 1991). The association between enrichment of a platelet phospholipid with n-3 fatty acids and a reduced risk of arterial thrombosis suggests that the accumulation of DHA in platelet phospholipids of modified egg consumers in this study (Table 3) may possibly be of some benefit. The significantly 0, I 0.01) elevated DHA level in platelet phospholipid of modified egg consumers may have resulted from the desaturation and elongation within the body of dietary LnA to DHA (Tinoco, 1982). Alternatively, it may have been due to the uptake of dietary DHA directly, since modified egg yolk had a significantly (p I 0.01) higher DHA level as compared to control egg yolk. Consumption of linseed oil (high in LnA, but devoid of DHA) failed to consistently change platelet phospholipid in human subjects (Sanders & Roshanai, 1983). The elevated DHA and not only LnA in the modified eggs may have been responsible for this rise in platelet DHA observed in this study. Other studies have failed to show a demonstrable rise in DHA in human platelet phospholipid despite a high intake of LnA at a low ratio (3/l) of n-6/n-3 fatty acid because of the limited metabolic conversion of LnA to DHA (Weaver et al., 1990). Despite the very high LnA content of the modified eggs, platelet phospholipid LnA levels remained extremely low following LNA-enriched egg consumption (see Table 3). Ingestion of linseed oil also failed to increase the level of LnA in platelet phospholipid to any appreciable extent (Sanders & Roshanai, 1983). Possible reasons for the low LnA level in platelet phospholipid may be the rapid P-oxidation of dietary LnA and subsequent metabolism including esterification into various tissue triglyceride pools. The EPA level within platelet phospholipid was unchanged following modified egg consumption which probably reflected the very minimal presence of EPA in the modified egg. The slight rise in docosapentaenoic acid (22: 5 n-3) in the platelet phospholipid may reflect some retroconversion of DHA. No changes were observed in platelet n-6 fatty acid levels following LnA-enriched egg consumption. In contrast, decreases in platelet phospholipid n-6 fatty

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L. K. Ferrier, L. Caston, S. Leeson, E. J, Squires, B. Celi, L. Thomas, B. J. Holub

acids have been documented following the ingestion of fish oil containing EPA and DHA (Weaver & Holub, 1988). In summary, consumption of LnA-enriched eggs resulted in a reduction in serum triglyceride levels and an accumulation of n-3 fatty acid, particularly DHA, in platelet phospholipid. These serum and platelet lipid alterations may possibly have an overall favourable effect on the risk of cardiovascular disease, but further studies are required to evaluate this potential. Also, the modified egg can provide a novel source of the essential n-3 fatty acids and availability of DHA for membrane phospholipid assembly and functioning.

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ACKNOWLEDGEMENT The authors appreciate the financial support of Maple Lynn Foods, the Ontario Egg Producers’ Marketing Board, and the Ontario Ministry of Agriculture and Food.

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