WO2005063230A1 - Use of conjugated linolenic acids for reducing body fat mass - Google Patents

Abstract

The present invention discloses the use of conjugated linolenic acids selected from the groups consisting of Cl 8:3 (9cis, 11 trans, 15 cis)octadecatrienoic acid, C18:3(9cis,13trans,15cis)octadecatrienoic acid and mixtures thereof in the reduction of body fat mass and in the treatment and/or prevention of obesity. These conjugated linolenic acids may be provided in the form of a food supplement or as a component of a prepared food product.

Description

USE OF CONJUGATED LINOLENIC ACIDS FOR REDUCING BODY FAT MASS

FIELD OF THE INVENTION

This invention relates to the use of conjugated linolenic acids to induce body fat reduction, thereby providing a treatment for diseases caused by or associated with an accumulation or increased level of body fat mass.

BACKGROUND OF THE INVENTION

Conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA).

Conjugated linoleic acid (CLA) is a general term used to name positional and geometric isomers of linoleic acid C18:2(9c/s,12c/s). It usually denotes a mixture of mainly two isomers: C18:2(9c/s, 11-rans) and C18:2(10-ra/ιs,12c/s), obtained by conjugation of linoleic acid C18:2(9c/s, 12cis) present as a major component in different vegetal oils (i.e. sunflower, safflower, soy, corn, etc.). They are usually present in a 1 :1 ratio and the sum of these two isomers can vary between 30 % and 90 %. The majority of CLA in nutraceutical market do not mention the accurate composition for the content of each isomer, but generally the product is around 80% for both isomers. Among the various isomers of CLA, c/s-9,frat7S-11- octadecadienoic acid (c9f11) is assumed to be the bioactive agent because of its abundance in natural food products (Chin et al. 1992; Ha et al. 1989; Parodi, 1997; Sehat ef a/. 1998a).

The free, naturally occurring CLA have been previously isolated from fried meats by Ha et al., (1987). Since then, they have been found in some processed cheese products (Ha et al., 1987).

Conjugated linolenic acid (CLNA) is a general designation for fatty acids with 18 carbons and three doubles bonds (C18:3 it is said that it is a trienoic fatty acid). CLA and CLNA are naturally formed in ruminant animals by biohydrogenation from the rumen bacteria Butyrivibrio fibrisolvens (Kepler et a/., 1967; Parodi, 1997; Destaillats et al., 2003). CLNA is naturally present as a minor component of cheese from cow milk (Winkler et al., 2001) and bovine milk fat (Destaillats et al., 2003). CLNA also occurs abundantly in some specific seed oils, such as kareia oil (Dhar et al., 1998), tung oil (Igarashi et al., 2000b) and pomegranate oil (Suzuki et al., 2001).

Natural fully conjugated linolenic acids have been found at high content levels in some seed oils (Hopkins, ln:Gunstone, F.D. (Ed), Topics in Lipid Chemistry, volume 3, ELEK Science, London, pp 37-87 [1972]).

Takagi and Itabashi (Lipids 16, 546 [1981]) reported 62.2% of calendic acid C18:3(8frans,10fraπs,12c/s) in pot marigold seed oil, 83.0% of punicic acid C18:3(9c/s,11 rat7s,13c/s) in pomegranate seed oil, 67.7% of α-eleostearic acid C18:3(9c/s, 11 trans, IStrans) in tung and 56.2% in bitter gourd seed oils, and 42.3% of catalpic acid C18:3 (9-rat7s,11-ra/7S,13c/s), in catalpa seed oil.

An octadecatrienoic acid isomer whose structure has been tentatively defined as C18:3 (9c/s,11 raπs,15c/s) is believed to be the first intermediate in the biohydrogenation process of α-linolenic acid by the anaerobic rumen bacterium Butyrvibrio fibrisolvens (C. R. Kepler and S. B. Tove 242 J. Biol. Chem. (1967) 5686).

Canadian patent application no. 2,396,840 discloses a particular mixture of isomers of the following conjugated linolenic acids: C18:3(9c/s,11 fraπs,15c/s) and C18:3(9c/^'s,13fraπs,15c/s). This mixture is commercialised under the trademark CLnA™. Both isomers are present in a 1 :1 ratio and their sum may vary between 35 % and 90 % depending on the degree of purification.

The resemblance between the most important isomer of CLA [C18:2(9c/s, 11 trans)] and one of the isomers of CLnA™ [C18:3(9c/^'s,11 frans,15cs)] in term of their structure is the 9c/s,1 λtrans insaturation. One can say that this isomer has a "CLA characteristic". The major difference between both isomers is the third insaturation: 15c/s. This insaturation confers a "omega-3 fatty acid characteristic". This should increase the bioavaibility of the product and therefore increase the activity of CLnA™. Current studies demonstrate the additive effects of these two characteristics (CLA and omega-3 fatty acid in the same molecule).

Recent studies have shown that conjugated trienoic fatty acids reduce body fat, modulate triacylglycerols metabolism and serum and liver lipid levels (free fatty acids, cholesterol and lipoproteins) in rats (Koba et al., 2002). In their study, rats feed with CLNA without specific identification of individual components resulted in a net reduction in adipose tissue weight. This effect was more potent than the one obtained with CLA. The adipose tissue weight-reducing effect of conjugated trienoic fatty acids may be related to a decrease either in triacylglycerol deposition from the circulation and / or in lipolysis in adipose tissues (Koba et al., 2002). Although the underlying mechanism is not clear at the moment, these findings indicate that CLNA modulate the body fat and^" triacylglycerols metabolism differently from CLA in rats.

Obesity

More than half of U.S. adults are overweight and nearly one-quarter of the U.S. adults are considered to be obese. Americans and Canadians spend more than $35 billion a year to lose weight and are getting heavier. The increasing prevalence of overweight and obesity is a major public health concern, since obesity is associated with several chronic diseases. For example, overweight and obesity are known risk factors for diabetes, heart disease, stroke and hypertension. Some people respond well to proper diet and exercise to overcome obesity. For those who do not, however, antiobesity drugs are gaining popularity in pharmaceutical companies. Drugs currently approved by the FDA for the treatment of obesity produce weight losses of about 10 % of initial body weight in one year when used singly. Caffeine and amphetamine based diet aids may be addictive and adversely affect other areas of health. Combination therapy with phentermine and fenfluramine produced weight losses of about 15 % of initial body weight in one year. Phenylpropanolamine is an over-the-counter drug that has not been tested for long term use and is recommended for use for only about 12 weeks. With the exception of phenylpropanolamine, all of these drugs require a physician's prescription and are generally quite expensive. Side effects occur with all these drugs. For example, the administration of fenfluramine and phentermine for the treatment of obesity resulted in cardiac valve damage in some patients and ultimately led to the withdrawal of fenfluramine from the market. Two of the newest drugs for the treatment of obesity have side effects that limit their use. Sibutramine increases blood pressure in a subset of patients, and orlistat may have unpleasant gastrointestinal side effects. Other dietary aids are available over the counter. Almost all of these chemical remedies have as their objective, weight loss through reduced food intake, brought about by appetite suppression.

CLA is best known for helping to reduce body fat and increase lean muscle mass which naturally slims the human body. It has been shown that CLA reduce adipose tissue and increase muscle (Delany et al. 2000; Thorn et al. 2001). In a recent clinical trial (Gaullier et al. 2004), overweight subjects (51 women and 10 men) taking CLA for one year, without changing their diet and exercise habits, had a 9 % reduction in body fat mass (BFM) and a 2 % increase in lean body mass (LBM) compared to the placebo group. In a short-term study, healthy subjects of normal weight experienced a 20 % reduction in body fat mass when CLA was combined with 90 min of exercise 3 times a week (Thorn et al., 2001). Additional studies lasted from 1 to 6 months and included men, women, healthy and / or overweight individuals. The results demonstrated a decrease in body fat mass with CLA. Riserus et al., (2001) demonstrated that CLA supplementation for 4 weeks in obese men may decrease abdominal fat, without concomitant effects on overall obesity or other cardiovascular risk factors. In their study, 14 abdominally obese men received CLA for 4 weeks and there was a significant decrease in abdominal diameter. Similar result were also obtained by others studies that concluded that CLA may reduced the proportion of body fat in human, by affecting the metabolism of fatty acids (Blankson et al. , 2000, Smedman et al., 2001). Effects of CLA on body composition have been documented in several animal models. CLA has been shown to reduce body fat and to increase lean body mass in pigs (Dugan et al. 1997), mice (Pariza et al. 1996; Park et al. 1997), rats and chicks (Pariza et al. 1996). In many animals models, CLA induce a decrease of adipose tissue, suggesting a possible implication in the clinical treatment of obesity (West et al. 1998; Ostrowska et al. 1999). One of the biological effects of CLA relates to fat accretion and nutrient partitioning. CLA has been shown to improve feed efficiency in rats, mice and chickens (Chin et al. 1994; Park et al. 1997) and to decrease carcass fat content in mice and rats (West et al. 1998; Sisk et al. 2001 ; Yamasaki et al. 2003). West et al. (1998) demonstrated that CLA reduced energy intake, growth rate, adipose depot weight, and carcass lipid and protein content. CLA significantly increased metabolic rate and decreased the night time respiratory quotient. Terspstra et al. (2002) showed that the decrease in body fat in mice fed CLA was due to increases in energy expenditure and energy loss in the excreta (Terpstra et al. 2002). Szymczyk et al. (2001) study the effects of CLA on growth performance, feed conversion efficiency and carcass quality in chickens. They found that CLA fat deposition was reduced. Feeding CLA to chickens resulted in a incorporation of CLA into their tissue lipids, thus providing a potential CLA source for human consumption.

The effect on body composition has been also demonstrated in pigs in several studies. Pigs fed 1 % dietary CLA had reduced feed intakes, improved feed efficiencies, deposited less subcutaneous fat, and gained more lean (Dugan et al. 1997; Cook et al. 1998). Backfat thickness in the CLA-fed pigs was reduced as early as day 14, reaching a 24 % decrease at the time of slaughter. Recent results, in pigs, suggest that dietary CLA supplementation reduced back fat thickness (Thiel et al. 1998) and the fat content of commercial meat cuts (Dugan et al. 1997). Ostrawa et al. (1999) found that dietary CLA increased the gain to feed ratio and lean tissue deposition and decreased fat deposition in finisher pigs. Wiegand et al. (2001) demonstrated that CLA supplementation improves feed efficiency, decreases backfat, and improves pork quality attributes of marbling and firmness of the longissimus muscle. Sensory characteristics were not different with CLA supplementation for tenderness, juiciness or flavor intensity.

CLA's have also been found to exert a profound generalized effect on body composition, in particular, upon redirecting the partitioning of fat and lean tissue mass. Cooks et al. in U.S. Pat. Nos 5,851 ,572; 6,020,378 and 6,060,087 disclose the use of CLA's for reducing body fat and increasing lean body mass. The method of treating meat animals to increase fat firmness, shelf life and meat quality indices consisting of administering to the meat animals a safe and effective amount of conjugated linoleic acid or CLA.

Cook et al., in U.S. Pat. No. 5,814,663 discloses the use of CLA's to maintain an existing level of body fat or body weight in humans. Remmereit et al., in U.S. Pat. No. 6,034,132 relates to a method for reducing body weight and treating obesity. The method comprises administering a nutritionally effective amount of conjugated linoleic acid to a human. The conjugated linoleic acid may be provided in the form of a free fatty acid in a pill, or as a component of a prepared food product.

Cook, et al. in U.S. Pat. No. 5,554,646 discloses a method utilizing CLA as a dietary supplement in which pigs, mice, and humans were fed diets containing 0.5 % CLA. In each species, a significant drop in fat content was observed with a concomitant increase in protein mass. It is interesting that in these animals, increasing the fatty acid content of the diet by addition of CLA resulted in no increase in body weight, but was associated with a redistribution of fat and lean within the body. Another dietary phenomenon of interest is the effect of CLA supplementation on feed conversion. U.S. Pat. No. 5,428,072 (Cook, et al.), provided data showing that incorporation of CLA into animal feed (birds and mammals) increased the efficiency of feed conversion leading to greater weight gain in the CLA supplemented birds and mammals.

Cook et al., in U.S. Pat. No. 5,760,082 relates to a dietetic food which contains a safe and effective amount of conjugated linoleic acid (CLA). Alviar et al. in U.S. Pat. No. 6,413,545 relates to a diet composition for managing body weight including effective amounts of CLA among others. Sagel et al. in U.S. Pat. No. 6,635,015 relates to body weight management systems for subjects including humans and domestic animals. Such systems utilize devices and compositions to shift the energy balance of the user in the direction wherein the calories burned due to the user's activity is greater than the calories consumed by that user. Such systems have a variety of uses including, but not limited to body weight maintenance, reduction or gain; reduction of body fat and, or gain in muscle mass and improvement of a subject's fitness. Sae butted-slashed, et al. in U.S. Pat. No 6,015,833 and Fimreite et al. in U.S. Pat. No. 6,524,527 both relate to novel compositions containing conjugated linoleic acids which are effective as animal feed additives and human dietary supplements.

Sugano et al., in U.S. Pat. No. 6,451 ,336 relates to an agent for increasing brown fat, comprising a conjugated linoleic acid as an active ingredient. More particularly, this patent relates to an agent for increasing brown fat, comprising a conjugated linoleic acid as an active ingredient, which agent can increase brown fat cells capable of consuming extra energy and producing heat to prevent obesity, and use of the agent in the field of foods.

McCleary et al., in U.S. Pat. No. 6,579,866 relates a nutritional supplement composition for modulating nutrient partitioning in a human so as to increase oxidation of fat and promote increased storage of glycogen. This supplement comprises CLA. The method of this invention results in marked increases in fat oxidation and glycogen storage while simultaneously minimizing fat synthesis and storage.

Cherwin et al., in U.S. Pat. No 6,124,486 relates to a process for making low calorie triglycerides having long and short fatty acid chains

U.S. Pat. Nos. 5,430,066 and 5,585,400 disclose the use of CLA to prevent weight loss due to immune stimulation and to treat immune hypersensitivity. Therefore, CLA may be used for increasing or maintaining weight gain in animals. Dietary supplementation with CLA results in weight loss in human patients. This is in contrast to studies disclosed in U.S. Pat. Nos. 5,428,072; 5,554,646 and 5,430,066 which disclose the dietary use of CLA to increase feed efficiency, increase weight gain, and maintain weight in the face of environmental challenges. From those studies, it could be predicted that dietary supplementation with CLA would increase the efficiency of food use resulting in weight gain or at least the maintenance of weight at a fixed level while body fat decreased.

Many other studies have been published on the potential of conjugated fatty acids (CLA) to modulate body fat mass and feed efficiency (Pariza, 2003; Azain, 2004; Ostrowska et al. 1999). Those effects have been design to be dependant of species, sex and genetic background of the animal. The sexes of the animals have been reported to be an important factor because females appeared to be more sensitive than males to CLA supplementation.

Among the CLA isomers, the 18:2 c-9,M 1 and 0,c-12 have been the most studied. The 18:2 M0,c-12 have been design to be the most effective for decreasing body fat mass (Pariza, 2003) while the 18:2 c-9,M 1 have been attributed to stimulate growth and increase feed efficiency in young rodents (Pariza, 2001). The mechanism for CLA efficiency may be the increase of adipocytes lipolysis with an increase of energetic expense which conducted to body fat diminution (West et al., 1998). Evans et al 2000 have also demonstrated that body mass effect may be due to apoptosis in preadipocyte cells.

The studies so far published in humans have not shown clearly the effect of CLA on body mass (Keim, 2003; Brugere et al, 2004). This may be due to different parameters like genetic background, BMI, sex, CLA doses and CLA isomers.

Omega-3 fatty acids and cardiovascular diseases

Comsumption of fish and fish oils was first associated with decreased risk of cardiovascular disease almost 50 years ago. Since then, a number of epidemiologic studies have evaluated whether their consumption is specifically associated with stroke. Studies have shown an inverse association between consumption of fish and fish oils and stroke risk. Omega-3 fatty acids come almost exclusively from fish oils. Currently, the effects of omega-3 fatty acids on reducing atherosclerosis, cerebrovascular accidents (CVA) and relapses after a first heart attack are well known. Omega-3 fatty acid supplementation reduces the risk of sudden cardiac death and death from any cause within 4 months in post- myocardial infarction patients (Harris et al. 2003). Evidence continues to accrue for benefits in the primary prevention of coronary heart disease and stroke. Several clinical studies have shown that regular consumption or supplementation in omega-3 fatty acids is beneficial for health (Marchioli et al. 2001 ; Iso et al. 2001 ; He et al. 2002; Skerrett et al. 2003). The cardioprotective effects of omega-3 fatty acids may be related to hypolipidemic, anti-thrombotic, anti-inflammatory, and anti- arrythmic functions (Harris et al., 2003). The evidence suggests that individuals with coronary artery disease may reduce their risk of sudden cardiac death by increasing their intake of omega-3 fatty acids

Researchers have hypothesized that recent changes in food sources have led to an imbalance in the optimal ratio of fatty acid intake. These imbalances may influence obesity. Specifically, modern diets have increased amounts of omega-6 fatty acids as compared to omega-3 fatty acids. Source of omega-3 fatty acids include conjugated linolenic acid like CLnA™ (Ha et al. 1987; Destaillats et al. 2003).

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the effects of CLnA ™ on glucose concentration in pig's blood

Figure 2 shows the effects of CLnA ™ on carbohydrates content in pig's carcass.

Figure 3 shows the interactions between different tissues in lipid and glucose metabolism. Figure 4 shows the effects of CLnA ™ on body weight development of male and female mice.

Figure 5 shows the effects of CLnA ™ on body composition of male and female mice. SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method of reducing body fat mass (BFM) in a subject.

It is also an object of the present invention to provide a method for the treatment or the prevention of disorders related and/or caused by an increased level of body fat mass, such as obesity.

Accordingly, the methods of the present invention comprise the step of administering to a subject an effective amount of a conjugated linolenic acid, said conjugated linolenic acid being an octadecatrienoic acid isomer selected from the group consisting of C18:3(9c/s,11 fraπs,15c/s), C18:3(9c/s,13-rans,15c/s) octadecatrienoic acid and a mixture thereof.

A further object of the present invention is to provide a functional food or a food supplement for a human or non-human animal which comprises a conjugated linolenic acid, said conjugated linolenic acid being an octadecatrienoic acid isomer selected from the group consisting of C18:3(9c/s,11-ra/?s,15c/s), C18:3(9c/s,13£rans,15c/s) octadecatrienoic acid and a mixture thereof.

Another object of the present invention is to use a conjugated linolenic acid for the manufacture of a composition useful for reducing body fat mass and/or treating or preventing disorders caused by an increased level of body fat mass, said conjugated linolenic acid being an octadecatrienoic acid isomer selected from the group consisting of C18:3(9c/s,11-rat7S,15c/s), C18:3(9c/^'s,13fraA?s,15c/s) octadecatrienoic acid and a mixture thereof. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery of the usefulness of conjugated linolenic acids in the induction of body weight reduction, and their use for preventing and/or treating disorders caused by an elevated level of body fat mass in a subject, such as obesity.

More specifically, the present invention is concerned with functional food, food supplements and methods for reducing body fat mass and for preventing and/or treating obesity in a subject.

As used herein, the term "subject" refers to any subject that is susceptible to have an elevated level of body fat mass. Among the subjects which are known to potentially have an elevated level of body fat mass, there are, but not limited to, humans and non-human animals such as farm animals, sport animals, zoological animals or wild animals.

A first aspect of the present invention concerns the use of a conjugated linolenic acid for the reduction of body fat mass in a subject, such as a human. According to a related aspect, the present invention provides a method of reducing body fat mass in a subject, said method comprising administering to said subject an effective amount of a conjugated linolenic acid.

The conjugated linolenic acids contemplated by the present invention is an octadecatrienoic acid isomer selected from the group consisting of C18:3(9c/s,11-ra/ιs,15c/^"s), C18:3(9c/s,13frans,15c/s) octadecatrienoic acid and a mixture thereof. It should be understood that in the context of the present invention, mono-, di- and triglycerol form, as well as, free fatty acids or any other ester form of the conjugated linolenic acids may be used. A preferred mixture of conjugated linolenic acids is CLnA™ commercialised by Naturia Inc. which is a 1 :1 mixture of C18:3 isomers: 9c/s,11frat7s,15c/s- octadecatrienoic acid and 9c/s,13fra/?s,15c/s-octadecatrienoic. Concentrations for the mixture may vary between 30% and 90%.

As one skilled in the art will understood, reduction of body fat mass leads to advantageously help treating and/or preventing a disorder caused by an elevated level of body fat mass in a subject, such as obesity. Such a skilled person is also aware that one of the problems associated with obesity is that even when excess weight is lost, such an effect is only temporary, and the individual regains weight soon after abandoning a diet or any other treatment method.

It is therefore another aspect of the invention to provide a method for treating and/or preventing obesity in a subject, said method comprising administering to the subject a therapeutically effective amount of a conjugated linolenic acid as defined above.

As used herein, the term "treating" refers to a process by which the symptoms of obesity in a subject are alleviated or completely eliminated. As used herein, the term "preventing" refers to a process by which obesity in a subject is obstructed or delayed.

According to a further aspect, the present invention is concerned with the use of a conjugated linolenic acid as defined above for the manufacture of a composition for reducing body fat mass in a subject, such as a human, or for treating and/or preventing obesity. Preferably, the conjugated linolenic acid is CLnA™. Such compositions contemplated by the present invention preferably take the form of food supplements or functional foods.

In this connection, a food supplement or a functional food according to the present invention comprises an effective amount of a conjugated linolenic acid as defined above. In the case of the food supplement, it is preferably provided in the form of a tablet, a capsule, a liquid or a powder. In the case of a prepared food product or functional food according to the present invention, it is meant any natural, processed, diet or non-diet food product to which, for instance CLnA™ has been added. CLnA™ may be added in the form of free fatty acids or as an oil containing partial or whole triglycerides of CLnA™. Therefore, CLnA™ may be directly incorporated into many prepared diet food products, including, but not limited to diet drinks and diet bars. Furthermore, CLnA™ may be incorporated into many prepared non-diet functional products, including, but not limited to candy, cookies, snack products such as chips, prepared meat products, milk, cheese, yogurt and any other fat or oil containing foods.

The amount of conjugated linolenic acids is preferably a therapeutically effective amount. A therapeutically effective amount of the conjugated linolenic acid is that amount necessary to allow the same to perform its body fat reduction role without causing, overly negative effects in the subject to which the conjugated linolenic acid is administered. The exact amount of conjugated linolenic acids to be used and administered will vary according to factors such as the type of obesity being treated as well as the mode of administration.

The conjugated linolenic acids or the compositions containing them may be given to a subject through various routes of administration. For instance, they may be administered in the form of sterile injectable preparations, such as sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art. The sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally- acceptable diluents or solvents. They may be given parenterally, for example intravenously, intramuscularly or sub-cutaneously by injection, by infusion or per os. Suitable dosages will vary, depending upon factors such as the amount of the conjugated linolenic acid in the composition, the desired effect (short or long term), the route of administration, the age and the weight of the subject to be treated.

In accordance with the treating and/or preventing method of the present invention, the conjugated linolenic acid as defined above (such as CLnA™) is preferably orally administered in a therapeutically effective amount to a subject, with obesity or not, or in a nutritionally effective amount to a subject of normal weight, to cause a loss of the desired body fat mass, and then continuously administered in a maintenance dose to prevent regaining the lost weight. It will be understood that a nutritionally effective amount is that amount of CLnA™ that, when ingested in purified form or as food supplement results in a reduction in body weight without impairing or interfering with proper nutrition. Typically, such amount may vary from 1 to 3 g a day. Accordingly, administration of a nutritionally effective amount of CLnA™ achieves weight loss without sensory deprivation associated with reduction in food intake. CLnA™ is useful to treat subjects with slight to profound clinical obesity. When treating subjects with clinical obesity, a therapeutically effective amount of CLnA™ which causes a reduction in weight of a clinically obese subject is administered. Typically, such amount may vary from 0.5 to 7 g a day.

In a typical regimen, an individual will begin the weight loss program by ingesting CLnA™ with each meal, and monitoring body fat mass. The CLnA™ may be provided in the form of a pill or as a component of a prepared food product contemplated by the present invention. Once the desired weight has been attained, a proper maintenance level can be found by gradually reducing the dose and continuing to monitor weight to assure there is no gain.

As one skilled in the art may appreciate, the use of a conjugated linolenic acid such as CLnA™ for the indications mentioned above is advantageous because CLnA™ is a non-toxic, naturally occurring food ingredient. CLnA™ is not classified as a drug and may be consumed as a part of a normal diet and finds use as a part of everyday nutrition.

EXAMPLES

The present invention will be more readily understood by referring to the following examples. These examples are illustrative of the wide range of applicability of the present invention and is not intended to limit its scope. Modifications and variations can be made therein without departing from the spirit and scope of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred methods and materials are described.

EXAMPLE 1 : Effect of conjugated α-linolenic acid (CLnA) on feed intake, body composition and liver fatty acids in pigs

32 piglets (castrated male and/or female) were used in this experiment over three weeks period following weaning (±18 days old). The piglets were distributed in 8 experimental blocks which include a piglet for every treatment ( 4 ) corresponding to a selection within the same litter.

Within each block, the animals were assigned to the following treatments: 1) an Omega-3 control: Ω-3 2) an Omega-6 control: Ω-6 3) a supplement of CLA (1 % of the diet): CLA 4) a supplement of CLnA (1 % of the diet): CLnA

All the treatments had the same quantity of stearic, oleic and palmitic acid and so the sum of fatty acids C18:2 and C18:3 are equal between all treatments. This allowed to observe directly and exclusively the effect of CLnA C18:3 (9cis, 11 trans, 15cis and 9cis, 13trans, 15cis) independently of the concentration of the other fatty acids.

Table 1 :

* Complete composition of the diet accounts for 100 g {balance = 92,58 g of fibers, corn, vitamins, minerals, etc)

All the diets were formulated to be isoenergetic, isoproteic (aminoacids), isolipidic (fatty acid). The level of food was controlled by the use of the gastric tube technique (Cortamira et al, 1991) as described by Girard et al (2001). Commercial food for first age of hasty weaning was in liquid form (1 : 2) (dry food :water). Lipid composition for each treatment is presented in Table 1.

TOXICOLOGY

The animals were weighed at the beginning of the experiment (Day 1) then three times a week. Food was adjusted according to their profit of metabolic weight. During this period, there was no treatment effect on growth of piglets and on the overall bodyweight.

At the end of the experiment (Day 19), animals were killed and organs (heart, liver, lungs, spleen, kidneys, brain and gastro-intestinal system) as well as carcasses were weighted. The results do not show any difference between treatments, indicating that no harmful effect.

Metabolic profiles (biochemistry)

Day 1 : Blood samples were taken before the morning meal by jugular venepuncture as described by Girard et al. (1986) immediately after weaning (before gastric cannulation) for determination of fatty acids, insulin, C-peptide and leptin. These were also determined on day 8 and 15. Blood samples were also analyzed for several classic markers of porcine metabolic profile (glucose, urea, creatinine, cholesterol, triglycerides, bilirubine, AST (SGOT), ALT (SGPT), Alkaline phosphatase, Creatine Kinase (CK), GGT, Lactate Dehydrogenase (LDH), total proteins, albumin (A), globulin (G), ratio A/G, calcium, phosphorus, Na, K, Cl, CO₂, and anionic gap).

Preliminary results indicate that no treatment modified significantly the porcine metabolism, and that these treatments do not induced toxicity, during the period of the treatment.

GLUCOSE TOLERANCE

Day 10: A jugular catheter was installed according to the method using a non- surgical technique described by Matte (1999).

Day 16 and 18: Two tests of glucose tolerance were made after infusion administration (2 h) of glucose (intragastric or parenteral: 1 ,0 g/kg/h) in the fasting animal. Each animal received successively the two infusions types at two days interval. Repeated blood samples were performed at 0, 30, 60, 90, 120, 150, 180, 210 and 240 min post-bolus for determination of plasma insulin, C-peptide, leptin and glucose concentrations.

Parenteral infusion of glucose

Effect on insulin

The 4 treatments (Ω3, Ω6, CLnA and CLA) increased the levels of insulin. In each case, the maximal dose of insulin was obtained 30 min after the infusion of glucose and it returns to the basal level after approximately 150 - 180 min.

At 30 min, CLnA induced a maximal concentration of insulin of 281 pM (281 X 10^{" 12} M), compared to 335, 297 and 264 pM for CLA, Ω3 and Ω6 respectively (n=8).

At 60 min, CLnA induced a concentration of insulin of 217 pM, which is significantly lower than the treatment CLA (280 pM, P=0.04, n=8), and slightly lower than the treatment Ω3 (253 pM, P=0.12, n=8).

At 210 min, the CLnA induced a significantly lower level than the treatment Ω3 (47 vs 55 pM, P=0.03, n=8).

The total area under the curve (which represents the total quantity of insulin) induced by CLnA (518 pM / h) is slightly lower than the one induced by CLA (566 pM / h) (P=0.27, n=8).

Effect on C-peptide

Similar results were obtained with C-peptide. C-peptide is produced during the conversion of proinsulin in insulin. C-peptide is synthetized in equimolaire quantity of insulin by the cells of the islands of Langerhans but contrary to insulin, C- peptide undergoes less degradation in the liver. This measurement is considered as a better indicator of insulin secretion than insulin itself, because of its better stability to hepatic metabolism. All the treatments (Ω3, Ω6, CLnA and CLA) increased the concentrations of C- peptide, with a maximum value at 30 min and a return to the basal level after approximately 150 - 180 min.

At 30 min, CLnA induced a maximal concentration of C-peptide of 349 pM, (similar to the treatment Ω3, 348 pM), which is significantly lower than CLA (397 pM, P = 0.05 %, n = 8).

At 60 min, CLnA still induced a lower concentration of C-peptide, compared to CLA and Ω3 (297 pM for CLnA, vs 348 and 342 pM for Ω3 and CLA respectively, P = 0.09, n = 8).

At 180 min, CLnA induced a lower value of C-peptide than the treatment Ω3 (22 and 26 pM respectively, P = 0.14, n=8).

The total area under the curve (which represents the total quantity of C-peptide), induced by CLnA is 591 vs 636 pM / h for CLA (P=0.4, n=8 ).

Effect on glucose

All 4 treatments (Ω3, Ω6, CLnA and CLA) increased the levels of glucose. In each case, the maximal dose of glucose was obtained after 30 min of infusion of glucose.

At 30 min, CLnA induced a maximal glucose concentration of 11.5 mM, while value for CLA is 11.6 mM, Ω3 is 12.6 and Ω6 is 11.2 mM, n = 6). Throughout the infusion (120 min), there is balance between the entry of blood glucose and the absorption.

At 120 min, the infusion of glucose is ended and we observed in each treatment, a decrease of glucose and a return to the basal level after approximately 150 min. The insulin had its effect and provoked a decline of the rates of blood glucose, by accelerating the transport of the glucose into cells. At 150 min, the glucose induced by the treatment CLnA is significantly more higher compared to the treatment Ω6 (3.9 vs 3.4 mM, P=0.05 , n = 6). Similar results are also obtained after 180 min (4.9, 3.9 (P=0.09), 3.9 mM (P = 0.12), for CLnA, Ω6 and CLA respectively, n = 6).

The total area under the curve (which represents the total quantity of glucose), showed that CLnA induced a total area of 28.6 mM / h, similar to the area induced by the treatment Ω3 (28.6 mM / h) compared to 27.7 and 27.2 mM / h, for the CLA and Ω6 respectively (n = 6).

Gastric infusion

Effect on insulin

The effect of CLnA on the levels of insulin was more pronounced after gastric administration than parenteral. The effect of CLnA on the levels of insulin was also more pronounced than that induced by the other treatments.

At 30 min, CLnA tended to increase insulin (297 pM, which is 40 % higher compared to CLA (P=0.13, compared with 274, 237 and 212 pM for Ω3, Ω6 and CLA respectively, n = 8).

The CLnA induced the same effect after 120 min, compared to CLA (130 and 100 pM, for the CLnA and CLA respectively, P = 0.11 , n = 8).

The total area under the curve is also slightly higher, 478 pM / h for CLnA, (compared to 442, 453 and 426 pM / h for CLA, Ω3, Ω6 respectively, n = 8).

Effect on C-peptide

The effect of CLnA on the levels of C-peptide was more pronounced after gastric administration than parenteral.

At 30 min, CLnA induced an increase of C-peptide of 343 pM (compared to 283, 301 and 270 pM for Ω3, Ω6 and CLA respectively, n = 8). At 120 min, CLnA induced an increase of C-peptide of 160 pM compared to 103 pM for the CLA (P = 0.07, n = 8).

On the other hand, after 210 min, CLnA induced lower levels of C-peptide compared to CLA ( 9 vs 29 pM respectively, P = 0.14, n = 8).

The total area under the curve is 589 pM / h (vs 472, 479 and 502 pM / h for the CLA, Ω3, Ω6 respectively, n = 6), which is a 20 % increase compared to CLA (P=0.10).

Effect on glucose

During the administration of infusion of glucose by gastro-enteral routes, the 4 treatments (Ω3, Ω6, CLnA and CLA) all increased the levels of glucose. The maximal dose of glucose was obtained after 30 min of glucose infusion. All the treatments induced an increase of glucose, which is lower compared to parenteral infusion.

At 30 min, CLnA induced an increase of 8.2 mM glucose, similar to Ω3 (compared to 7.6 and 7.5 mM for Ω6 and CLA respectively, n=6). Throughout the infusion (120 min), there was a balance between the entry of blood glucose and the absorption.

At 120 min, the infusion of glucose is ended. CLnA induced a significant increase of glucose, compared to CLA (8.2 vs 6.8 mM, P = 0.01 , n = 6). This increase of glucose is significantly higher than the treatment Ω6 (7.1 mM, P = 0.04, n = 6). After 120 min, CLnA had also increased the levels of insulin.

The inventors observed, at approximately 180 min, a decrease of glucose and a return to the basal level. At 180 min, the levels of glucose induced by CLnA are 5.1 mM compared to 4.3 mM for CLA (P = 0.14, n = 6).

The total area under the curve, showed that CLnA induced a slightly higher area than that induced by CLA (24.1 vs 22.7 mM / h, P = 0.10, n = 6) and Ω6 (22.9 mM / h, P=0.14, n = 6). BODY COMPOSITION

After 2 weeks of different treatments (Ω3, Ω6, CLnA and CLA), piglets were killed (Day 19), and the carcasses were frozen and crushed, to estimate the total contents of water, ash, proteins, fat and carbohydrates.

The body composition indicate that CLnA treatment acts on the metabolism. The water content (humidity) do not indicate significant variations between different treatments.

The CLnA, as well as Ω3, decreases the levels of proteins (16.5 and 16.2 % for CLnA and Ω3 respectively) compared to treatments Ω6 and CLA (17.1 and 17.7 % for Ω6 and CLA respectively).

Also, the levels of fat in the carcass is lower after CLnA treatment (8.8 %, compared to 8.9 % for both Ω3 and CLA and 9.3 % for Ω6).

By calculating the levels of carbohydrates (subtraction), the inventors noticed that in the CLnA treatment these levels were increased.

These results showed that CLnA modified the body composition in agreement with the previous results on glucose tolerance.

CONCLUSIONS

The effects of CLnA™ were more pronounced after gastric administration than with CLA. These were related to glycaemia while the effects of CLA are more related to insulin, indicating a novel and unique mechanism of action.

Two different parameters suggest that the Omega-3 characteristic of CLnA™ is active in glucose metabolism: glucose concentration in blood and carbohydrates content in carcass. a) Glucose concentration in blood: For CLnA™ and Omega-3 (OME), glucose circulates longer than CLA and Omega-6 (W6) in the blood stream, allowing to burn off the circulating glucose as fuel instead of stocking it as fat (Figure 1). b) Carbohydrates in carcass: Carbohydrates content in carcass showed that with CLnA™ and Omega-3, glucose is preferably stored as glycogen form in muscles (carcass; Pathway 1), indicating that it is less available for triglycerides conversion by the liver (Pathway 2) and subsequent storage as fat deposit. Pathways 1 and 2 are better illustrated in Figure 3, in which interactions between different tissues in lipid and glucose metabolism are shown.

EXAMPLE 2 : Effect of conjugated α-linolenic acid (CLnA) on feed intake, body composition and liver fatty acids in Mice

Various metabolic effects have been demonstrated following administration of conjugated linoleic acid (CLA), including changes in body composition in animals.

In this Example, male and female ICR mice were fed with 1% CLA isomer (cis-

9,trans- ), an equimolar CLnA mixture of two isomers (c/s-9,fraπs-11 , c/s-15 and c/s-9,-rans-13, c/s-15) or a control group (fatty acids from a mixture of high oleic sunflower oil/linseed oil 98:2 w/w) given as free fatty acids for 6 wk. Body fat mass was significantly reduced in females fed CLA or CLnA, with a greater extent with the CLnA diet. There was a significant interaction between sexes and diet for ash content only. No significant body composition modifications were observed for males. No changes in organ weight, empty carcases and feed efficiency were observed in males and females. From these data, the inventors conclude that CLnA is more effective than rumenic acid in body composition modifications in female.

The aim of this example was to investigate the potentiality of an equimolar mixture of conjugated α-linolenic acid (c/^'s-9, raπs-11 , c/s-15 and c/s-9, f/ans-13, c/s-15 18:3) to reduce body fat mass in mice and to compare it to the effect produced by c/s-9, trans λ 18:2 CLA isomer.

MATERIALS AND METHODS

Fatty acids

Free fatty acids of CLA were purchased at Natural ASA (Norway) and Conjugated linolenic acid (CLnA™) was prepared by Naturia Inc. (Sherbrooke, Canada). The high oleic sunflower oil was purchased at Lesieurs, (France) and the linseed oil at Robbe SA (France). A mixture of high oleic sunflower oil and linseed oil (98:2 W/W) was saponified to obtain free fatty acids for the control diet instead of the free fatty acids studied.

Animals

6 week-old males (n=27) and females (n=27) ICR mice were purchased from Elevage Janvier (Saint Genest, France). The initial weight of the animals was 32.18 ± 1.76 g for the males and 24.33 ± 0.77 g for the female respectively.

The mice were housed in pools of 3 animals in plastic cages in an animal house at constant temperature (22 ± 1 °C) and relative humidity (55-60%) with a 12-h light- dark cycle. They were adapted during 3 days with commercial pellets before being allocated to one of the 3 dietary groups (see below). 9 males and 9 females were allocated to each group. The average starting weight in each group for the male was 32.99 ± 0.04g for the control group, 32.73 ± 0.04g for the CLA group and 33.11 ± 0.04g for the CLnA group. The average starting weight in each group for the female was 25.01 ± 0.04g for the control group, 24.95 ± 0.04g for the CLA group and 25.11+ 0.04g for the CLnA group. Diets

The experimental diets were fed in pellets fabricated by INRA (Jouys-en-Josas, France). The diets contained (g/kg) wheat starch: 460, sucrose: 220, casein: 180, high oleic sunflower/linseed oils 98:2 w/w: 50, mineral mix: 50, cellulose: 20, Studied lipids: 10, vitamins mix: 10. The studied lipids were CLA and CLnA as FFA. The fatty acid content of each diet is summarized in Table 2. Feed was exchanged three times a week. At that time, the remaining pellets were removed and weighed to determine food intake. The animals were weighed two times a week.

Body composition, feed efficiency and lipid analyses

At the end of the 6 weeks experimental period, the animals were killed by decapitation. The liver, heart and gastrocnemian muscle were removed from the carcass, blotted and weighed. The livers were stored in a chloroform/methanol (2:1 v/v) solution at -20°C until lipid extraction. After removing gut, the empty carcass were weighed, frozen in liquid nitrogen and stored at -20°C before body composition determination. Body composition was determined in 6 males and 6 females per group. Briefly, protein content was calculated as nitrogen content multiplied by 6.25. Nitrogen content was measure by the Kjeldahl method. Total lipids were determined by the Folch method. Minerals were measured as ash content after incineration at 500°C for 6h. Feed efficiency was calculated from food consumption and weigh gain during 6 week experimental period. Liver lipids were extracted according to the procedure of Folch et al. The lipid content was determined by gravimetry. Liver lipids have been separated into phospholipids and neutral lipids as described by Juaneda and Rocquelin (1985). All the lipids classes have been methylated with sodium methylate (1N). The resulting FAME were then analysed by a gas-liquid chromatograph using a Hewlett-Packard serie II gas chromatograph packet with a BPX70 capillary column (SGE, Melbourne, Australia, 120m x 0.25 mm i.dx0.25 μm film thickness). FAME were identified using authentic standards. CLA metabolites were identified as described by Sebedio et al. (1997) and CLnA metabolites were identified as described by Destaillats et al. (2004). Quantitative data were obtained using the Diamir software (JMBS Developments, Le Fontanil, France).

Statistical analysis

Data are presented as mean ± SD using SigmaStat software as a two ways (diet and gender) ANOVA procedure. Post hoc analyses were performed using the Newman-Keul test. P values of less than 0.05 were considered as significant.

RESULTS

Body weight

Figure 4 shows mean body weight development during experimental period. Body weight increased from 33 g to 41 g in males and from 25 g to 29 g for females. No significant difference was observed between the experimental groups. Weight gain ranged between 3.6 g and 15.0 g in males and 1.2 and 12.0 in females.

Feed efficiency

Feed efficiency (expressed as gain/feeds) is presented in Table 3. There was a significant difference between males and females but not among the three diets.

Empty carcass and organ weight

Table 4 summarizes the weight of the empty carcasses and the different organs studied. The empty carcass weight ranged between 24.8 and 26.2 for males and 17.5 to 18.2 for females respectively without any significant differences between groups. The weight of the other organ studied was not significantly different among groups. Body composition

Figure 5 shows the body composition of the mice after the 6 weeks experimental period. A diminution of body fat mass was observed in the female group fed CLnA. However, the difference is not significant (P 0.17) due to large inter group differences. The differences between male and female fed CLnA was not significant (P=0.09). However, strong tendency seems to guide the body fat pattern of female fed CLnA.

Fatty acids profile in liver

Fatty acid profile of liver PL was modified by the ingestion of CLA and CLnA and is presented in Table 5. An increase of palmitic acid was observed in males fed CLA and CLnA compared to control group. This increase was compensated by a significant diminution of oleic acid into those groups but also into females fed CLnA. Also, arachidonic acid (20:4 n-6) was decreased in males fed CLnA and also in female fed CLA compared to control group. CLnA diet decreased the incorporation of DHA (22:6 n-3) fatty acid compared to control diet.

The fatty acid composition of liver TAG was slightly modified by the ingested CLA and CLnA as seen in Table 6. However, cholesteryl esters (Table 7) were modified with large intra group variability which could hide some effects of feeding CLA and CLnA. For instance, palmitic acid in female fed CLA and CLnA is not significant even with important differences between the two groups.

CONCLUSION

CLnA feeding of both isomers decreased body fat mass (p=0.17) in females compared to control group. A slight effect was observed in males. REFERENCES

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Table 2: Fatty acid composition (%) of the lipid fraction of each diet

Table 3 : Feed efficiency of male and female mice fed control diet, CLA or CLnA diets. Results are expressed as means (SD) of 9 independent determinations

"Values are different between males and females

Table 4 : Empty carcass and organ weight (g)

Organ/Diet

Males* Females Males Females Males Females

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

Heart 0,198 0,034 0,159 0,020 0,198 0,018 0,151 0,032 0,185 0,023 0,151 0,039

Liver 2,04 0,28 1,45 0,34 2,24 0,29 1,43 0,27 2,09 0,45 1,43 0,19

Gastrocn. 0,117 0,040 0,091 0,031 0,135 0,040 0,109 0,021 0,145 0,033 0,113 0,029

Empty carcasses 26,2 4,1 18,2 3,1 24,8 2,3 17,5 2,0 26,6 3,4 17,6 1 ,5 values are different between males and females

Table 5: Fatty acid composition (%) of liver phospholipids of male and female mice fed CLnA, CLA or control diet. Results are expressed means (SD) of 9 independent determinations.

Control CLA CLnA

Males Femelles Males Femelles Males Femelles

Fatty acids Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE

Saturated

14:0 0,02^a* 0,01 0,15 0,04 0,17"* 0,07 0,26 0,09 0,25" 0,13 0,24 0,05

16:0 17,97^a* 2,33 24,06 3,26 23,70" 3,74 26,65 2,47 28,27" 3,97 24,36 2,82

18:0 17,03 1,26 17,07 2,32 16,10 1,41 16,84 2,57 14,96 2,09 17,82 2,18

Monounsaturated

16:1n-9 0,25^a* 0,06 0,43 0,07 0,47" 0,08 0,45 0,11 0,49" 0,06 0,45 0,05

16:1n-7 1,21^a 0,31 1,39 0,34 1,62^ac 0,17 1,81 0,51 2,15^b 0,62 1,55 0,51

18:1n-9 19,37^a* 1,10 17,21^a 0,83 16,80" 0,93 16,72^ac 0,86 16,28" 0,96 15,58"^c 1,09

18:1n-7 4,45 1,33 3,31 0,78 3,79 0,75 3,92 0,84 3,85 0,76 3,96 1,01

Polyunsatu rated

18:2n-6 10,38 1,40 10,14 1,07 11,27 0,80 11,03 1,07 11,90 2,09 10,54 0,64

c-9,M1 18:2 ND ND ND ND 0,86 0,30 0,77 0,26 ND ND ND ND

c-9,.-11,c-15 + c-9,t ND ND ND ND ND ND ND ND 0,46 0,11 0,40 0,07

13,c-15 18:3

20:3n-6 2,70^a* 0,55 1,82 0,24 2,15"* 0,39 1,42 0,20 2,10" 0,37 1,68 0,21

20:4n-6 14,01^a 1,56 13,43^a 1,37 13,00^ac 1,87 10,83" 1,40 11,34 ^c 2,31 13,59^a 1,48

22:5n-3 0,19^a* 0,05 0,09 0,02 0,12"* 0,02 0,07 0,02 0,09" 0,01 0,07 0,02

22:6n-3 5,90^a 1,06 6,14^a 1,25 4,72^ac 1,25 4,55^b,= 0,94 3,60^bc* 0,84 5,56^ac 1,13

ND = not detected

Values with the same letter are not significant

Male values with an * are different from female

Table 6: Fatty acid composition (%) of liver cholesterol esters of male and female mice fed CLnA, CLA or control diet. Results are express as means (SD) of 9 independent determinations.

Control CLA CLnA

Males Femelles Males Femelles Males Femelles

Fatty acids Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE

Saturated

14:0 0,09^a* 0,07 0,27^a 0,02 0,31" 0,04 0,18^ac 0,25 0,13" 0,09 0,061^b,= 0,03

16:0 14,81 5,17 18,80^a 9,31 14,69 5,65 11,26^ac 8,27 10,14 2,05 7,22"^c 1,51

18:0 4,87^a 1,61 3,68 1,20 3,65^ac 0,56 3,22 1,28 2,97"^c 0,65 3,00 0,59

Monounsaturated

16:1n-9 0,56^b,= 0,17 0,93 0,17 1,34^a* 0,27 0,78 0,48 0,90^ac 0,27 0,47 0,10

16:1n-7 4,39*"= 1,80 6,48 2,43 9,36^a* 2,65 3,27 3,13 8,32^ac 2,40 4,65 1,40

18:1n-9 60,65 6,88 59,90^a 9,33 56,79 6,15 63,86^a,= 4,91 63,43 4,15 71,92^bc 1,89

18:1n-7 5,21 1,22 3,51 1,24 4,14 0,93 5,30 1,28 5,11 1,30 5,12 1,60

Polyunsaturated

18:2n-6 4,09* 1,52 2,63 0,39 2,72 1,06 2,56 0,62 3,74 0,84 2,84 0,39

c-9,M1 18:2 ND ND ND ND 3,38 0,50 4,36 2,51 ND ND ND ND

c-9,f-11,c-15 + c-9,t-13,c- ND ND ND ND ND ND ND ND 0,93 0,40 1,36 0,33 15 18:3

20:4n-6 2,11" 0,77 1,25 0,39 1,00^bc 0,57 1,01 0,81 1,67^ac 0,72 1,31 0,38

22:6n-3 0,72 0,33 0,67 0,31 0,32 0,15 0,83 0,92 0,60 0,25 0,56 0,19

ND = not detected

Values with the same letter are not significant

Male values with an * are different from female

Table 7: Fatty acid composition (%) of liver triacylglycerols of male and female mice fed CLnA, CLA or control diet. Results are expressed means (SD) of 9 independent determinations.

Control CLA CLnA

Males Femelles Males Femelles Males Femelles

Fatty acids Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE

Saturated

14:0 2,12^ac 0,41 1,45 0,40 1,42^a 0,40 1,44 0,65 2,36^bc 0,54 1,64 0,46

16:0 33,22 3,74 27,11 3,69 30,85 5,31 28,45 8,26 31,29 3,84 28,81 8,09

18:0 1,13^a 0,24 1,40 0,50 1,99^b 0,84 1,61 0,61 1,17^a 0,28 1,45 0,22

Monounsaturated

16:1n-9 2,53 0,54 2,51 0,66 2,96 0,73 2,38 0,93 2,87 0,57 2,46 0,58

16:1 n-7 6,96^a 1,79 4,78 1,63 4,71 ^,= 1,25 6,16 1,97 6,15^ac 1,30 4,86 1,03

18:1n-9 44,59* 4,52 55,43 4,73 47,40 4,19 49,72 5,84 45,02 3,67 51,59 7,21

18:1 n-7 5,04 1,23 3,65 1,00 4,72 0,85 3,98 0,87 5,15 0,94 4,30 1,29

Polyunsaturated

18:2n-6 2,64 0,97 2,62 1,01 2,38 1,24 2,23 1,00 2,79 1,39 2,35 1,04

C-9.M118:2 ND ND ND ND 1,48 0,82 2,50 1,98 ND ND ND ND

C-9,.-11,C-15 + C-9,t-13,C- ND ND ND ND ND ND ND ND 1,28 0,39 1,49 0,27

1518:3

20:4n-6 0,14 0, 12 0,09 0,08 0,14 0,20 0,25 0,31 0,10 0,06 0,08 0,04

22:6n-3 0,03 0,02 0,03 0,03 0,03 0,04 0,08 0,12 0,02 0,02 0,02 0,01

ND = not detected

Values with the same letter are not significant

Male values with an * are different from female

Claims

CLAIMS:

1. A method of reducing body fat mass in a subject, said method comprising administering to said subject an effective amount of a conjugated linolenic acid, said conjugated linolenic acid being an octadecatrienoic acid isomer selected from the group consisting of C18:3(9c/s,11 ra/7S,15c/s), C18:3(9c/^'s,13.ra/7S,15c/s) octadecatrienoic acid and a mixture thereof.

2. A method for treating and/or preventing obesity in a subject, said method comprising administering to the subject a therapeutically effective amount of a conjugated linolenic acid, said conjugated linolenic acid being an octadecatrienoic acid isomer selected from the group consisting of C18:3(9c/s,11 rat7s,15c/s), C18:3(9c/s,13fraπs,15c/s) octadecatrienoic acid and a mixture thereof.

3. The method of claim 1 or 2, wherein said subject is a human.

4. The method of claim 1 or 2, wherein said subject is a non-human animal.

5. A functional food for a human or non-human animal, comprising an effective amount of a conjugated linolenic acid, said conjugated linolenic acid being an octadecatrienoic acid isomer selected from the group consisting of C18:3(9c/^'s,11 fraπs,15c/s), C18:3(9c/s,13fraA?s,15c/s) octadecatrienoic acid and a mixture thereof.

6. A food supplement for a human or non-human animal comprising a conjugated linolenic acid, said conjugated linolenic acid being an octadecatrienoic acid isomer selected from the group consisting of C18:3(9c/^'s,11.ra/7s,15c/s), C18:3(9c/s,13-rat7s,15c/s) octadecatrienoic acid and a mixture thereof.

7. The food supplement of claim 6, wherein it is in the form of a tablet, capsule, liquid or powder.

8. Use of a conjugated linolenic acid for the reduction of body fat mass in a subject, said conjugated linolenic acid being an octadecatrienoic acid isomer selected from the group consisting of C18:3(9c/s,11-rans,15c/s), C18:3(9c/s,13frat7s,15c/s) octadecatrienoic acid and a mixture thereof.

9. Use of a conjugated linolenic acid for the treatment and/or prevention of obesity in a subject, said conjugated linolenic acid being an octadecatrienoic acid isomer selected from the group consisting of C18:3(9c/s,11 raπs,15c/s), C18:3(9c/s,13.ra/7s,15c/s) octadecatrienoic acid and a mixture thereof.

10. The use of claim 8 or 9, wherein the subject is a human.

11. The use of claim 8 or 9, wherein the subject is a non-human animal.

12. Use of a conjugated linolenic acid for the manufacture of a composition for reducing body fat mass in a subject, said conjugated linolenic acid being an octadecatrienoic acid isomer selected from the group consisting of C18:3(9c/s,11 frat7S,15c/s), C18:3(9c/s,13fraπs,15c/^'s) octadecatrienoic acid and a mixture thereof.

13. Use of a conjugated linolenic acid for the manufacture of a composition for treating and/or preventing obesity, said conjugated linolenic acid being an octadecatrienoic acid isomer selected from the group consisting of C18:3(9c/s,11fraA?s,15c/s), C18:3(9c/s,13-rat7S,15c/s) octadecatrienoic acid and a mixture thereof.