Research and Reviews: Dairy

Special Circular 163-99

Conversion of Dietary trans₁₁ 18:1 to Conjugated Linoleic Acid in Mice

J. E. Santora1
D. L. Palmquist
The Ohio State University
Department of Animal Sciences

K. L. Roehrig
The Ohio State University
Department of Food Science and Technology

2 For more information, contact at: The Ohio State University, Ohio Agricultural Research and Development Center, 312 Gerlaugh Hall, 1680 Madison Avenue, Wooster, OH 44691; 330-263-3795; fax: 330-263-3949; e-mail: palmquist.1@osu.edu

Abstract

Conjugated linoleic acid (CLA) has been associated with many health benefits such as inhibiting cancer. Trans fatty acids have been associated with health risks such as atherosclerosis. Researchers have suggested that trans11 18:1 can be converted to CLA by the action of the enzyme D9 desaturase. Two studies were conducted to quantify the conversion of dietary trans11 18:1 to conjugated linoleic acid (CLA) and to determine metabolic modifiers that may influence the conversion in mice. Approximately 50% of the stored trans11 18:1 was desaturated to CLA in both experiments. Clofibrate, used to induce activity of the enzyme, did not increase conversion of trans11 18:1 to CLA; however, the conversion was reduced by a diet containing high amounts of polyunsaturated fatty acids (PUFA). Desaturation of dietary trans11 18:1 is an important source of CLA for the body. Since both trans11 18:1 and CLA are found in milk fat, data from this research show that milk fat is an important contributor to a healthy diet.

Introduction

Milk fat contains a fatty acid popularly known as CLA. CLA is a collective term for isomers of linoleic acid that have been shown to have potent anticarcinogenic effects, especially in breast cancer (Ip et al., 1991). In addition to cancer prevention, CLA may contribute to antiatherogenesis, reduction of body fat, promotion of lean muscle mass, stimulation of immune functions, and enhancement of bone formation (Doyle, 1998). The CLA content of milk fat for dairy cows has been reported to range from 2 to 30 mg/g with variation attributed to seasonal influences on pasture conditions and variations in fat intake and rumen function (Parodi, 1977). The most important dietary source of CLA is milk fat, but it can also be found in the fat of meat from other ruminants (beef, lamb) (Parodi, 1994, 1997). Although the human dietary requirement of CLA is unknown, extrapolation from animal studies suggests that human dietary intake may be less than optimal for achieving maximum anticarcinogenic protection.

Scientists are studying ways to increase CLA content of the diet. Though CLA content in milk fat can be increased by selective feeding of the cow, this is associated with an increase in trans11 18:1 in the milk fat. Trans fatty acids in milk are normally about 2% of total fatty acids, but can increase to as much as 5-10% (Hunter and Applewhite, 1986). Trans fatty acids are regarded as unfavorable for both dairy cows and humans. Trans fatty acids occur in human diets in the form of margarine, vegetable oils, dairy products, beef, and lamb. The trans fatty acids in margarine and other vegetable oils are formed by chemical hydrogenation; although there can be many trans isomers, trans9 predominates in these. CLA and the trans11 18:1 isomer in animal products are formed through microbial biohydrogenation of dietary unsaturated fatty acids in the rumen (Harfoot and Hazlewood, 1988). Researchers have suggested that trans11 18:1 can be converted by D9 desaturation to CLA (Hohman and Mahfouz, 1981). This conversion may be one way to maximize CLA available to the body.

The objectives of the present studies were to quantify the conversion of dietary trans11 18:1 to CLA in the whole animal and to determine whether conversion of trans11 18:1 to CLA may be influenced by metabolic modifiers. Trans11 18:1 was fed to mice at 1% of the diet; conversion was determined by measuring the total CLA in the body after a two-week feeding period. In addition, metabolic modifiers were fed in the second study to influence activity of the desaturase enzyme that converts trans11 18:1 to CLA (normal, induced, or inhibited enzyme activity).

Materials and Methods

Experiment 1

Animals

Eighteen female mice, six to seven weeks old, were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, Ind.). Sixteen of the mice were individually housed in plastic cages with wire mesh bottoms and were allotted randomly to four replicates of four treatments. Mice were given free access to water and fed a semi-purified powdered basal diet containing either 0 or 1% trans11 18:1 (Table 1). Half the mice were fed for ad libitum intake (nibblers). The other half of the mice were trained to eat their food within two hours once a day (meal-fed) to stimulate fatty acid synthesis and enzyme activity. The remaining two mice were used to determine initial body composition and content of trans11 18:1 and CLA.

Experiment 2

Animals

Thirty-five female mice, six to seven weeks old, were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, Ind.). Thirty of the mice were individually housed in plastic cages with wire mesh bottoms and were allotted randomly to five replicates of six treatments - two levels of trans11 18:1 (0 or 1%) and three levels of enzyme activity (normal, induced, or inhibited) in a 2 x 3 factorial arrangement of treatments (Table 1). Mice were given free access to water and fed for ad libitum intake. Clofibrate (0.5% of the diet) was used to induce desaturase activity. Polyunsaturated fatty acids (PUFA) fed at 10% of the diet in the form of corn oil was used to inhibit desaturase activity. The five remaining mice were used to determine initial body composition and content of trans11 18:1 and CLA.

Measures

Body weights and food offered and refused were measured daily. At the end of the two-week feeding period, all mice were sacrificed and contents of the cecum and stomach were removed to obtain empty carcass weight. The carcasses were freeze dried, chopped, and ground in a blender with liquid nitrogen, and the dry weights were determined. Fatty acid content and profiles of the carcasses were determined.

Statistical Analysis

Treatment differences were analyzed by a general linear model for main effects (diet and feeding protocol) and interactions (diet x feeding protocol) for Experiment 1. For Experiment 2, treatment differences were analyzed by a general linear model for main effects (diet and modifiers) and interactions (diet x modifiers).

Results and Discussion

Experiment 1

Though fatty acid synthesis and desaturase activity is increased in meal-fed rats, meal-fed mice were not capable of maintaining adequate food intake to gain body weight and store fat; therefore, data are not presented for this treatment group.

Although not significant, animals fed trans11 18:1 ate less than animals not fed trans11 18:1 (Table 2), thus fat intake and energy intake tended also to be lower. Total trans11 18:1 intake was as expected; animals fed trans11 18:1 consumed 494 mg of trans11 18:1, and animals not fed trans11 18:1 did not consume trans11 18:1.

Body weight (data not shown) and fat content were not different between 0 and 1% TFA dietary groups (Table 3). However, fat content mass and as a percentage of carcass were slightly lower in animals fed trans11 18:1, which is consistent with observations that CLA intake reduces body fat content (West et al., 1997). Trans11 18:1 was not detectable in animals not fed trans11 18:1. The CLA content and percentage of carcass in mice fed trans11 18:1 were higher than in mice not fed trans11 18:1.

Experiment 2

The main effect of trans11 18:1 on food intake was similar to Experiment 1; animals that were fed trans11 18:1 ate less than animals not fed trans11 18:1 (P < 0.05), and total fat intake and energy intake also were lower in animals fed trans11 18:1 (Table 4). Total trans11 18:1 intake was as expected; animals consumed 460 mg of trans11 18:1 whereas animals not fed trans11 18:1 did not consume trans11 18:1. Animals fed metabolic modifiers (clofibrate and PUFA) ate less (P < 0.05) than animals not fed a modifier (Table 5). The PUFA group ate significantly less than the control animals. Total energy intake per metabolic body size (kcal day-1/kg BW0.75) was lower in animals fed trans11 18:1. The PUFA significantly decreased (P < 0.05) energy intake of mice fed trans11 18:1.

Body weights (data not shown) and fat contents were not different among groups; however, fat contents and fat percentages tended to be lower (P < 0.03) for groups fed modifiers (Table 6). Animals fed trans11 18:1 stored 39.9 mg more of trans11 18:1 in their bodies than animals not fed trans11 18:1. The clofibrate and PUFA groups stored less trans11 18:1 than the control group (Table 7). The CLA content and percentage in the carcass increased (P < 0.05) in animals fed trans11 18:1. The clofibrate and PUFA groups stored less CLA than the control group (P < 0.05).

The net gains of CLA were 38.8 mg and 39.9 mg for Experiments 1 and 2, respectively, which represents the difference between body CLA in mice fed trans11 18:1 and body CLA in mice not fed trans11 18:1 (Table 8). The conversion of dietary trans11 18:1 was 7.8% and 8.7%, which represents the amount of body CLA expressed as a percent of trans11 18:1 consumed {[Carcass CLA (treatment - control)/dietary trans11 18:1] x 100}. However, interpretation of the conversion of dietary trans11 18:1 is limited because most of the trans11 18:1 was either oxidized or otherwise lost from the body. The conversion of stored trans11 18:1 may be a more useful measure; thus we present the amount of CLA found in the body as a proportion of trans11 18:1 available for conversion {[Carcass CLA (treatment - control)/carcass CLA + trans11 18:1 (treatment - control)] x 100}. Of the amount of trans11 18:1 that was available for incorporation into the body tissues, 48.4% was desaturated to CLA in both Experiments 1 and 2. Although desaturation was not increased by clofibrate, the conversion was reduced 30% by increased dietary PUFA.

To study in detail the conversion of trans11 18:1 to CLA, we chose to examine CLA accumulation in the body in relation to the amount of trans11 18:1 stored in tissues and therefore available for conversion. This storage amounted to less than 10% of the trans11 18:1 consumed, a retention that is apparently lower than for most other dietary fatty acids. Yet, because trans11 18:1 is found in milk fat in consistently higher amounts than is CLA, usually 5-10% times as much, the trans fatty acid in milk fat is an important contributor to total CLA in the body. Indeed, we found that supplementing 1% trans11 18:1 in the diet tripled the amount of CLA found in the carcass of the mouse. In this context, the recent Finnish studies are significant, where it was found that including a large amount of hydrogenated vegetable oil in the diet of human volunteers increased CLA in plasma lipids by 30% (Salminen et al., 1998), and infusing trans11 18:1 into the intestine of cows increased CLA content of the milk fat by 50% (Griinari, 1998). These two related studies support the hypothesis that trans11 18:1 is converted to CLA by desaturation; our study is the first to quantitatively measure the conversion of trans11 18:1 in vivo.

Trans fatty acids in the diet are viewed negatively by public-health advocates. Our work helps to distinguish among the positional isomers of the trans fatty acids. Trans11 18:1, the predominant trans fatty acid found in ruminant fat, is an important contributor to the body's supply of the important anticarcinogen, CLA.

Conclusions

The D11 isomer of trans monoene fatty acids may be considered a health benefit rather than a health risk because it contributes to the body's supply of CLA. Since milk fat contains both trans11 18:1 and CLA, the dairy industry may add another claim to their "Drink milk, it does a body good" campaign.

References

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Salminen, I., M. Mutanen, M. Jauhiainen, and A. Aro. 1998. Dietary trans fatty acids increase conjugated linoleic acid levels in human serum. J. Nutr. Biochem. 9:93-98.

West, D. B., P. M. Camet, C. D. Maddux, J. Scimeca, and J. P. DeLany. 1997. Reduced body fat with conjugated linoleic acid feeding in the mouse. FASEB J. 11:A599.