J. Agric. Food Chem. 2002, 50, 4941−4946
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Differential Incorporation of Conjugated Linoleic Acid Isomers into Egg Yolk Lipids LIN YANG,†,‡ YU HUANG,§ ANTHONY E. JAMES,| LIK WANG LAM,| ZHEN-YU CHEN*,‡
AND
Department of Chemistry, Henan Normal University, Henan, China; Department of Biochemistry, Department of Physiology, and Laboratory Animal Service Centre, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
The incorporation pattern of conjugated linoleic acids (CLA) isomers into the egg yolk of hens in relation to that in the diet was studied. Silver-ion high-performance liquid chromatography (Ag-HPLC) was used to separate individual CLA isomers. It was found that the isomeric distribution pattern in the egg yolk lipids was different from that in the dietary fat. Total cis/trans isomers accounted for 81.2% of total CLA incorporated into the egg yolk, which was in contrast to the value of 92.0% of total CLA in the diet. Total cis/cis isomers accounted for 3.8% total CLA in the diet but they were 6.6% of the total CLA in the egg yolk lipids. In contrast, total trans/trans isomers were 12.2% of the total CLA isomers in the egg yolk lipids, whereas they were only 4.2% of total CLA in the diet. The results showed that total trans/trans-CLA was preferentially incorporated into the egg yolk, whereas the incorporation of total cis/trans-CLA isomers was partially discriminated. Within each group, the incorporation of individual isomers into the egg yolk lipids was also selective. cis-9,trans-11/trans9,cis-11 and cis-10,trans-12/trans-10,cis-12 were the two major isomers in the diet. Ag-HPLC analysis showed that the former was preferentially transferred into the egg yolk compared with the latter. It was observed that supplementation of CLA in the diet of laying hens decreased the concentration of oleic acid (18:1n-9), arachidonic acid (20:4n-6), and docosahexaenoic acid (22:6n-3) but increased that of linolenic acid (18:3n-3), stearic acid (18:0), and palmitic acid (16:0) in the egg yolk, suggesting that CLA may inhibit 46 and 49 desaturases. KEYWORDS: Conjugated linoleic acids; egg; hens; phospholipids; triglycerides
INTRODUCTION
Conjugated linoleic acids (CLA) are a group of positional and geometric isomers of octadecadienoic acid. Various animal models have demonstrated that CLA are anticarcinogenic (1), hypolipidemic (2), and antiatherosclerotic (3-4). CLA have also been shown to enhance immune functions (5-6) and reduce fat accumulation while they increase muscle and bone mass (78). CLA could behave like an antioxidant in some systems (910), but be a prooxidant in others (11-12). CLA in food supply are quantitatively minor, and hence their consumption in humans is only 0.5-1 g/d/person (13). In addition to taking CLA supplements, feeding animals with a synthetic CLA mixture should be an alternative to enrich CLA in foods. Supplementation of CLA in the diet of laying hens has led to incorporation of CLA into eggs and changes in yolk * Corresponding author (telephone (852) 2609-6382; fax (852) 26035123; e-mail
[email protected]). † Henan Normal University. ‡ Department of Biochemistry, The Chinese University of Hong Kong. § Department of Physiology, The Chinese University of Hong Kong. | Laboratory Animal Service Centre, The Chinese University of Hong Kong.
fatty acid composition (14-20). Although the efforts were given to separate individual CLA isomers of egg yolk lipids in previous studies (14-20), quantification of each isomer incorporated using gas-liquid chromatography (GLC) was impossible because of poor resolution and overlap of CLA isomers. Silver-ion high-performance liquid chromatography (Ag-HPLC) has been shown to resolve CLA isomers well and demonstrated that CLA mixture contained at least 12 isomers (21, 22). Using the Ag-HPLC technique, the present study was carried out further to monitor the incorporation of each CLA isomer into the egg yolk lipids and to ascertain whether there is a selective transfer of CLA isomers. MATERIALS AND METHODS Diets. Two CLA mixtures were obtained as gifts from Natural Lipids Ltd, AS, Norway (mixture A) and Bioriginal Food & Science Corp., Saskatoon, SK, Canada (mixture B). The CLA blend supplemented in the diet was a mixture of A and B in a ratio of 1.7 to 1 (wt/wt). A basal chicken diet named poultry breeder was purchased from Glen Forrest Stockfeeds (Western Australia, Australia). According to the supplier, the diet contained 16.0% protein, 4.2% fat, 5.3% fiber, 3.5% calcium, 0.6% phosphorus, 0.3% sodium chloride, and varying amounts
10.1021/jf020363d CCC: $22.00 © 2002 American Chemical Society Published on Web 07/17/2002
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J. Agric. Food Chem., Vol. 50, No. 17, 2002
Yang et al.
Figure 1. Typical silver-ion high-performance liquid chromatograms of conjugated linoleic acids (CLA), as a form of methyl esters, derived from (A) diet
and (B) egg yolk lipids. c, cis; t, trans. of vitamins and other minerals. The control diet was prepared by adding 5% canola oil into the basal diet, whereas the CLA diet was formulated by adding 5% canola oil and 2.2% CLA blend. It was expected that 5% CLA would incorporate into the total egg yolk lipids at 2.2% CLA supplementation in diet according to Du et al. (20). The total lipids from both the control and CLA diet were extracted using chloroform and methanol (2:1, vol/vol), and their fatty acid composition was analyzed as described below. Hens. Fifteen CSIRO Hybrid White Leghorn (Gallus domesticus, 20 wks) were divided into two groups and housed (5/cage) in a room at 25 °C with a 12-h light/dark cycle. The first group (n ) 5) was fed the control diet, whereas the second group (n ) 10) was fed the CLA diet. The diets were ad libitum given to the hens, and uneaten food was discarded daily. Eggs were collected daily for a period of 4 weeks. The protocol was reviewed and approved by the Committee of Animal Ethics, The Chinese University of Hong Kong. Fatty Acid Analysis. The diets, 3-5 eggs from the control group, and 7-10 eggs from the CLA group collected on days 0, 6, 11, 16, and 28 were analyzed for their fatty acid composition. Egg yolk was separated from the egg white, and the total lipids derived from the egg yolk were extracted using chloroform/methanol (2:1, vol/vol) containing triheptadecanoic acid as an internal standard. To determine the egg yolk fatty acid composition, acid-catalyzed methylation was used. In brief, the egg yolk lipids (20 mg) were transesterified in 2 mL of 14% BF3 in methanol under nitrogen at 95 °C for 2 min. Hexane (4 mL) and distilled water (3 mL) were then added and mixed thoroughly. After the mixture was centrifuged, the top hexane layer containing fatty acid methyl esters (FAME) was saved and subjected to gas-liquid
chromatographic (GLC) analysis. It was found that the intra-isomerization among the CLA isomers was minimal (
