# ASPECTS TECHNOLOGIQUES ET DEVELOPPEMENT-ASPECTS TECHNOLOGIQUES ET DEVELOPPEMENT

## Technological and development aspects Combining linoleic acid and functional food?

Oléagineux, Corps Gras, Lipides. Volume 8, Numéro 4, 328-32, Juillet - Août 2001, Dossier : "Aliments fonctionnels et lipides"

**Auteur(s)** : Jean-Louis SEBEDIO, Lionel BRETILLON, Jean-Michel CHARDIGNY, Inra, Unité de nutrition lipidique, 17, rue Sully, BP 86510, 21065 Dijon Cedex, France.

**Résumé** : L'acide linoléique conjugué (CLA) est présent dans les aliments d'origine animale, en particulier ceux issus des ruminants. Le CLA peut aussi être synthétisé au laboratoire par isomérisation alcaline de l'acide linoléique. Alors que l'isomère naturel est le 18 : 2 9c,11t, les produits de synthèse contiennent principalement deux isomères, les 18 : 2 9c,11t et 10t,12c. De nombreuses études menées sur des modèles animaux, le plus souvent avec des mélanges synthétiques, montrent des effets potentiellement bénéfiques pour la santé (anticarcinogènes, protecteurs vis-à-vis de l'athérosclérose, modulateurs de la composition corporelle...) alors que la plupart des études menées chez l'homme concernent les effets des CLA sur la composition corporelle. À ce jour, les données concernant les effets sur la composition corporelle chez l'homme semblent indiquer un bénéfice potentiel, mais les résultats restent controversés. Par ailleurs, bien qu'un effet anticarcinogène soit démontré chez l'animal, aucune étude chez l'homme n'a pu établir de relation entre CLA et cancer du sein. De plus, peu d'études traitant des aspects sécuritaires liés à la consommation à long terme ont été publiées. Par conséquent, il est nécessaire de disposer de plus de données en particulier chez l'Homme pour garantir l'innocuité de ces acides gras afin de protéger le consommateur, avant d'envisager l'utilisation de CLA dans des aliments fonctionnels.

**Mots-clés** : acide linoléique conjugué, aliments fonctionnels, cancer, composition corporelle, athérosclérose, immunité, sécurité alimentaire.

**Summary** : Conjugated linoleic acid (CLA) is found naturally in many animal products especially those from ruminant sources. CLA can also be synthesized in the laboratory by isomerisation of linoleic acid. While the "natural" isomer is the 9c,11t-, synthetic mixtures contain mainly two isomers, the 9c,11t- and the 10t,12c-18:2. Many experiments carried out on animal models mainly using the synthetic isomers have shown different health benefits (anticarcinogenic, antiatherosclerotic effects, modulation of body composition...) while most of the studies carried out on human concern the effects of CLA on body composition. At the moment, the data on the effects on body composition in human seem to indicate a potential benefit but are still controversial. Furthermore even if anticarcinogenic effects have been demonstrated in animals, studies so far carried out on human have failed to demonstrate a relation between CLA and breast cancer and very few data dealing with
the safety of using CLA in long term feeding studies have been so far published. Consequently, before
using CLA in functional food, it will be necessary to obtain more human data and to be able to
guarantee the safety of these fatty acids in order to protect the consumer.

**Keywords :** conjugated linoleic acid, functional food, cancer, body composition, atherosclerosis, immunity, food safety.

ARTICLE

Due to their physiological effects, mainly demonstrated in animals, conjugated linoleic acid (CLA) has been stimulated research for the past few years.

CLA is mainly found in dairy products and meat from ruminants [1, 2]. In this case, the 9c,11t- isomer represents 85-90% of the total CLA isomers [3, 4]. It is produced by microbial conversion in the rumen and delta9 desaturation of vaccenic acid in the mammary gland of lactating cows [5].

However, CLA mixtures have also lately being produced by the industry [6]. The starting material in most cases is linoleic acid or oils rich in linoleic acid (sunflower, safflower). In general, the fatty acids are converted into conjugated forms with strong alkali [7]. In this case, a mixture of isomers is obtained in which the 10t,12c- and the 9c,11t- each represents about 40% of the total CLA isomers. These are accompanied by other ct/tc isomers (8,10; 11,13).

So far, many studies have been carried out using these "synthetic mixtures", which are now well defined considering the development of analytical techniques based on the combination of gas liquid chromatography, and silver-nitrate high performance liquid chromatography [3, 8].

This paper will review the research so far carried out on CLA and the potentialities of enriching foodstuff in CLA and/or using CLA in functional food.

Potential beneficial effects of CLA in animals

CLA and cancer prevention

Prevention of cancer by CLA in animals is with certainty the field of research which has been the most deeply investigated since the early 1990s. We do not aim to review exhaustively all the experiments carried out on CLA and cancer, extensive papers have already been published so far [9, 10]. Rather we aim to give in this section a brief general overview on CLA and mammary cancer prevention in animals.

The role of CLA was evaluated *in vitro* and *in vivo* in rat or mouse models of chemically-induced cancers of the mammary gland, of forestomach, intestine, prostate and skin. Concerning mammary carcinogenesis, a typical experiment follows a design in which CLA is added to the diet during gland maturation (52d post-wean-ing in the rat), the carcinogen is then added and tumor progression is analyzed in term of number and size of tumors and tumor incidence along the following weeks. Two carcinogens were used by Ip and co-workers in 1994 [11]: 7,12-dimethylbenz(a)anthracene (DMBA) or methylnitrosourea (MNU). CLA given to rats at 1% of the diet was able to reduce the number of tumors and tumor incidence by about 35% in both chemically-induced models. This finding was of
importance as it suggests that CLA would reduce tumorigenesis by interfering in the metabolism of DMBA and/or either by preventing the direct alkylating action of MNU on DNA. While this study was carried out with a mixture of CLA consisting of 9c,11t-18:2, 10t,12c-18:2 in equal proportion and other minor isomers, one other trial was performed with rumenic acid (9c,11t-18:2). Compared to the CLA mixture, rumenic acid was as effective to reduce the number and incidence of mammary tumors [12] (Table). How do CLA act? One mechanism would be by reducing the mass of the mammary epithelium and the terminal end bud density in the mammary gland [12]. In such a way, the maturation of the mammary epithelium would be down-regulated, and would explain the CLA-induced reduction of mammary cancer.

## CLA and body composition

CLA exhibit beneficial effects on lean to fat mass composition in animals. CLA-induced reduction of fat to protein mass was reported to occur in rats [13], mice [14-19] or pigs [20, 21]. Again more or less complex mixtures of CLA isomers were used for these experiments. However, the 10t,12c-18:2 isomer was consistently associated with this effect [14, 15]. The mechanism that underlies this action may be found in the dual effect of CLA on carnitine palmitoyltransferase and lipoprotein lipase activities. By enhancing the former enzyme in muscle and inhibiting the latter in adipose tissue [17], CLA may reduce the intracellular fatty acid concentration, and as a consequence would explain the reduction of fat mass.

CLA also exhibit one other interesting action in connection with growth. Animals fed with CLA showed significant reduction of feed intake without affecting consistently body weight [14, 18]. This would result from a better feed efficiency.

## CLA, blood lipids and atherosclerosis

So far few studies have evaluated the role of the different CLA isomers taken alone on blood lipids. Nevertheless, the lipid lowering effect of CLA mixtures [22, 23] can be assigned to 10t,12c-18:2, but not to 9c,11t-18:2 [22, 24]. In this field, assuming that the reduction of cholesterol and triacylglycerols levels in blood lipids would represent a less favourable atherosclerotic profile, dietary CLA may thus prevent such a disease in animals. However, caution should be taken when looking at the results of a study performed in C57/BL6 mice by Munday *et al.* [25]. Mice were fed 15 weeks an atherogenic diet containing 10% of cholesterol and 2.5 or 5% of CLA. Serum triacylglycerol level was lowered and the ratio of HDL- to total cholesterol was increased in the CLA treated mice. These data are consistent with the results reported above and in-line with the correlation between CLA consumption and a less atherogenic profile. However, aortic fatty streak area has also been analyzed, and these authors reported increased values in mice fed 2.5% of CLA. One may thus contradict the potential benefits of CLA in connection with the blood lipids, if not considering the great variability of the data within the different groups: 0.13 ± 0.13mm² in the control group, 0.33 ± 0.27mm² in the 2.5%-CLA group (mean ± SD). In view to extrapolate the effects observed in animals to humans, other models which physiology is more comparable to humans, rabbits and pigs were investigated. Rabbits fed daily 0.5g of CLA and a pro-atherogenic diet (0.1% cholesterol), showed a significant lower LDL- to HDL-cholesterol ratio after 22 weeks and thicker aortic fatty lesions, although the difference in this parameter failed to reach significance [26]. Later this study was re-evaluated to test whether CLA would prevent also regression of fatty streaks. At dietary levels of 0.1% CLA inhibited atherosclerosis in rabbits, and at 1% CLA substantially caused a 30% regression of established lesions [27]. Adult
female swines were fed 6 weeks a diet containing 1% of CLA (34.6% rumenic acid plus 9t,11c-18:2, 18.4% 10t,12c-18:2 and minor isomers). Compared to the control animals fed sunflower oil, triacylglycerol and cholesterol contents of VLDL and LDL were increased by CLA. The difference reached significance for the level of triacylglycerols in LDL fractions only (+58%) and for the 26% increase of the LDL- to HDL-cholesterol ratio [28]. This is in contrast to other animal studies since it would suggest a positive correlation between CLA consumption and atherogenesis.

## CLA and the immune response

During an immune stimulation macrophages are activated, leading to the production of cytokines like IL-1, and eicosanoids like prostaglandin E₂ from arachidonic acid. IL-1 induces PGE₂ release which in turn down regulates IL-1 production. Application of cytokines and PGE₂ to muscle leads to its degradation, called cachexia. When included in the diet, CLA prevented immune-induced weight loss [29]. This effect has positively been correlated to the inhibition of PGE₂ production in response to lipopolysaccharides in a macrophage cell line [30]. CLA has also been shown to enhance immune function by increasing circulating and splenic T-lymphocytes [31].

## Human studies

Only limited data are available on CLA supplementation in humans and most of the studies published are short term experiments dealing with the effect of CLA on body composition.

One study done in Norway [32] on overweight or obese volunteers using 3.4g of CLA for 12 weeks showed a reduction of mean body weight and body fat mass in the CLA group compared to the placebo group (olive oil), but the data were not significant. However, in another experiment, the same group [33] found a reduction of body fat in the CLA group when it was administered at 1.8g/day to healthy exercising humans for 12 weeks. In studies report-ed by Vessby et al. [34] using CLA (mixture of 9c,11t- and 10t,12c-18:2), or purified 10t,12c-18:2 at 3.4g/day for 3 months, indications of reduction of body fat in the CLA groups were found but the differences were of border line significance. However, while the CLA mixture did not affect serum lipids or glucose metabolism, the 10t,12c- isomer caused an "impairment of the peripheral insulin sensitivity, blood glucose concentrations and HDL levels in serum".

Studies carried out in USA also using a CLA mixture [35], showed that supplementation of CLA for 2 months at 3g/d did not affect body composition or energy expenditure in adult women. In a similar 9 weeks experiment Medina et al. [36] demonstrated that 3g of CLA mixture given to the same population decreased circulating leptin concentrations in the first part of the study without any detectable changes of body fat mass while it returned to baseline levels over the last two weeks of supplementation. Furthermore, supplementation did not affect appetite, and the authors concluded that "these results are counter to what would be expected if CLA were able to reduce body fat in humans as it does in animals".

Two other human studies looking at the effect of CLA on blood parameters (platelet function, blood coagulation) were published recently [37]. It looks like a short term (93d) consumption of CLA (mixture of isomers) did not induce any changes on blood coagulation and platelet function in healthy adult females. Furthermore, CLA administration for 63 days did not change the level of plasma cholesterol, low density or high density lipoprotein cholesterol and triglycerides. On the
contrary to animal studies previously described CLA did not seem to have any health benefits as far
as the prevention of atherosclerosis is concerned. However, we must be careful when drawing
conclusions as these are short term studies using a very complex mixture of isomers.

To our knowledge, only the results of one study dealing with breast cancer were recently reported and are available as an abstract [38]. While extensive data have demonstrated that CLA have anticarcinogenic properties in animals, no evidence was found to document the potential of CLA to influence breast cancer presentation at diagnosis or to decrease the risk of metastases after the treatment.

Two studies also reported the effect of CLA on the human immune status. One study (93d) done on young healthy women showed that CLA (complex mixture of isomers) did not have beneficial effect on their immune status [39]. On the contrary, a study carried out in the Netherlands showed that a specific mixture of CLA (50% 9c,11t-, 50% 10t,12c-) stimulates the humoral immune response as reflected by an increase of the seroprotection rate (SPR) while a 80/20 CLA mixture did not affect the SPR [40].

Safety aspects

Animal studies

Among the numerous studies reporting physiological effects of CLA in animals, only a few of them
are dealing with safety aspects. Only one experiment was conducted in order to study specific
toxicological effects of CLA supplementation on male Fischer 344 rats [41]. It was reported that
feeding CLA induced no modification of the organ weight, except thymus and no modification of the
haematological profile. However, some other papers report some data on safety, even if it was not
the major topic of the study.

Rat and mice studies

Most of the rat studies were carried out using the Sprague-Dawley strain. As previously described by Scimeca, several authors reported that CLA feeding did not induce any significant modifications of liver weight [13, 42, 43]. Sugano et al. [44] reported in 1997 that feeding CLA (1% in the diet) did not induce any modification of the weight of liver, heart, kidney, lung, spleen, brain and perirenal adipose tissue. In a further study [45], an increase of the liver weight (+9%) concomitantly with a decrease of the perirenal adipose tissue weight (-31%) was mentioned. No significant effects on the weight of other tissues were described. An increase in the liver lipid content has also been reported [46], but only after feeding 1.5% CLA. In the same study, Moya-Camarena et al. observed a decrease of the liver lipid content in female mice (but not in male) after feeding 0.5% CLA. Lipid droplets in the liver tissues were also observed by Yamasaki et al. [47] after feeding 2% CLA.

Stangl et al. [23] reported that feeding CLA up to 5% in the diet did not induce any modification of plasma protein, urea, ASAT and ALAT. However, albumin and creatinine levels were increased after 5 weeks of a diet containing 5% of CLA.

Some studies reported an enlargement of the liver [18, 25, 48], together with a modification of the colour of the organ and a hepatic lipidosis. These results were confirmed by Tsuboyama-Kasaoka et al. [19] who observed a strong decrease of the adipose tissues (white and brown) weights
concomitantly with an enlargement and lipidosis of the hepatic tissue. However, these data along
with those of Munday *et al.*, were obtained with C57/BL6 mice. It seems that this strain of mice is
more sensitive to the dietary treatment. Feeding AKR/J mice for 39d with 1% CLA-supplemented
diets, resulted in an increase of liver (+28%) and spleen (+18%) weight, but no lipid accumulation was
observed in the hepatic tissue [16].

Besides these effects on the liver size, Belury's group [49] also reported a peroxisome proliferator like response, as CLA feeding induced an increase in ACO-, CYP4A1- and FABP-mRNA. On the other hand, Jones *et al*. concluded that such effects were not observable in rats, suggesting a specie-dependent response [50].

Other animal studies

CLA treatment also were evaluated on pigs and hamsters. F1B hamsters have been shown to present
an increase in liver and kidney weight [24] after CLA supplementation. However, the kidney was
heavier only after supplementation with the 10t,12c-CLA isomer. This point is important as all the
other studies were carried out using CLA mixtures from different origins, which means different CLA
isomer profiles. In the same study, the 10t,12c-isomer was also reported to induce a liver
hypertrophy, as illustrated by a decrease of the number of nuclei in the histological analysis. On the
other hand, hamsters did not present a peroxisome proliferator-like response, as indicated by the
lack of increase of the related enzyme activities. Again, different responses may be observed
according to the animal species used. To our knowledge, only one study in pigs mentioned some
safety aspects. Feeding a CLA mixture for 6 weeks, Stangl *et al*. [28] reported no modification of T3
and T4, albumin, creatinine, urea and glucose in the plasma.

Taken all together, it is difficult to conclude about the safety of CLA supplementation, as
observations may vary according to the different species and strains of animals. Moreover, the
composition of the CLA mixtures used may also explain some differences. The results reported on
C57BL6 mice are the most interesting, but well-conducted safety studies according to international
guidelines are mandatory before taking any conclusions.

Human studies

Besides these animals studies, few human studies have also been so far reported and some of them
mentioned some safety aspects. However, all the published studies used CLA mixtures from
different origins. Consequently, final conclusions are difficult to draw as the effects of each isomer is
not reported.

We can mention here the publication from Basu *et al.* [51] who reported that feeding CLA (4.2g/d, 12wk) induced an increase in the lipid peroxidation status in men, as illustrated by the urinary isoprostane content. On the other hand, it is stated that "No participant experienced any side effects during the study period". It was also demonstrated [52] that even if CLA induce both non-enzymic and enzymic lipid peroxidation in men, the lipid peroxidation parameters had returned to their basal levels 2 weeks after stopping the CLA intake (Figure).
A human double-blind placebo-controlled safety study was recently reported [32]. The control subjects received olive oil where as the treated ones ingested CLA (3.4g/d, 12wk). No modification of blood parameters (blood lipids, hematology, liver enzymes, electrolytes, creatinine, LDH) were observed and the paper concluded on the safety of CLA under these experimental conditions.

Another human study was reported by Kelley *et al.* [39]. Young healthy women were fed 3.9g/d CLA for 63d. No modification of circulating white cells and no change in lymphocyte proliferation were observed. The latter differs from results obtained in different animal species, i.e. mice, rats and chicken [29, 53, 54].

These data seem to indicate that CLA supplementation is safe in humans, but long term studies with isolated isomers are needed before drawing definitive conclusions, as some data from animal studies indicate the CLA may have some adverse effects.

## CONCLUSION

From the data which have been so far published, we may conclude that the results obtained from the animal experiments are much more convincing than the human data. However, most human experiments have been short term studies, using complex mixtures of CLA isomers and we think that it is rather difficult to conclude at the moment. We need more human studies using supplementation with pure isomers and we also need more data on the safety of CLA supplementation on long term basis. At the moment our laboratory is in collaboration with AFSSA determining the CLA content of different food items sold on the french market in order to be able to evaluate the consumption of CLA by the consumer and to further carry out epidemiological studies. No doubt that all the studies which are planned in different laboratories working on CLA will permit to determine soon if utilisation of CLA in functional food is beneficial for the consumer.

## REFERENCES

1. FRITSCH E, FRITSCH J (1998). Occurrence of conjugated linoleic acid isomers in beef. *J Am Oil Chem Soc*, 75: 1449-51.

2. PARODI PW (1977). Conjugated octadecadienoic acids of milk fat. *J Dairy Sci*, 60: 1550-3.

3. SEHAT N, KRAMER JKG, MOSSOBA MM, *et al*. (1998). Identification of conjugated linoleic acid isomers in cheese by gas chromatography, silver ion high performance liquid chromatography and mass spectral reconstructed ion profiles. Comparison of chromatographic elution sequences. *Lipids*, 33: 963-71.

4. LAVILLONNIÈRE F, MARTIN JC, BOUGNOUX P, SÉBÉDIO JL (1998). Analysis of conjugated linoleic acid isomers and content in French cheeses. *J Am Oil Chem Soc*, 75: 343-52.

5. GRIINARI JM, CORL BA, LACY SH, CHOUINARD PY, NURMELA KVV, BAUMAN DE (2000). Conjugated linoleic acid is synthesized endogenously in lactating dairy cows by delta9-desaturase. *J Nutr*, 130: 2285-91.
6. GNÄDIG S, RICKERT R, SEBEDIO JL, STEINHART H (2001). Conjugated linoleic acid (CLA): physiological effects and production. *Eur J Lipid Sci Technol*, 103: 56-61.

7. REANEY MJT, LIU YD, WESCOTT ND (1999). Commercial production of conjugated linoleic acid. In: YURAWECZ MP, MOSSOBA MM, KRAMER JKG, PARIZA M, NELSON GJ, ed. *Advances in conjugated linoleic acid research*, volume I. Champaign, Illinois: AOCS Press, 39-54.

8. CHRISTIE W (2001). A practical guide to the analysis of conjugated linoleic acid. *INFORM*, 12: 147-52.

9. SCIMECA J (1999). Cancer inhibition in animals. In: YURAWECZ MP, MOSSOBA MM, KRAMER JKG, PARIZA M, NELSON GJ, ed. *Advances in conjugated linoleic acid research*, volume I. Champaign, Illinois: AOCS Press, 420-43.

10. KRITCHEVSKY D (2000). Antimutagenic and some other effects of conjugated linoleic acid. *Br J Nutr*, 83: 459-65.

11. IP C, SINGH H, THOMPSON HJ, SCIMECA J (1994). Conjugated linoleic acid suppresses mammary carcinogenesis and proliferative activity of the mammary gland in the rat. *Cancer Res*, 54: 1212-5.

12. IP C, BANNI S, ANGIONI E, et al. (1999). Conjugated linoleic acid-enriched butter fat alters mammary gland morphogenesis and reduces cancer risk in rats. *J Nutr*, 129: 2135-42.

13. STANGL GI (2000). Conjugated linoleic acids exhibit a strong fat-to-lean partitioning effect, reduce serum VLDL lipids and redistribute tissue lipids in food-restricted rats. *J Nutr*, 130: 1140-6.

14. PARK Y, ALBRIGHT KJ, STORKSON JM, LIU W, COOK ME, PARIZA MW (1999). Changes in body composition in mice during feeding and withdrawal of conjugated linoleic acid. *Lipids*, 34: 243-8.

15. PARK Y, STORKSON JM, ALBRIGHT KJ, LIU W, PARIZA MW (1999). Evidence that the trans-10,cis-12 isomer of conjugated linoleic acid induces body composition changes in mice. *Lipids*, 34: 235-41.

16. DELANY JP, BLOHM F, TRUETT AA, SCIMECA JA, WEST DB (1999). Conjugated linoleic acid rapidly reduces body fat content in mice without affecting energy intake. *Am J Physiol - Regulatory Integrative and Comparative Physiology*, 45: R1172-9.

17. PARK Y, ALBRIGHT KJ, LIU W, STORKSON JM, COOK ME, PARIZA MW (1997). Effect of conjugated linoleic acid on body composition in mice. *Lipids*, 32: 853-8.

18. WEST DB, DELANY JP, CAMET PM, BLOHM F, TRUETT AA, SCIMECA J (1998). Effects of conjugated linoleic acid on body fat and energy metabolism in the mouse. *Am J Physiol*, 275: R667-72.

19. TSUBOYAMA-KASAOKA N, TAKAHASHI M, TANEMURA K, et al. (2000). Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice. *Diabetes*, 49: 1534-42.
20. DUGAN MER, AALHUS JL, SCHAEFER AL, KRAMER JKG (1997). The effect of conjugated linoleic acid on fat to lean partitioning and feed conversion in pigs. *Can J Anim Sci*, **77**: 723-5.

21. OSTROWSKA E, MURALITHARAN M, CROSS RF, BAUMAN DE, DUNSHEA FR (1999). Dietary conjugated linoleic acids increase lean tissue and decrease fat deposition in growing pigs. *J Nutr*, **129**: 2037-42.

22. GAVINO VC, GAVINO G, LEBLANC MJ, TUCHWEBER N (2000). An isomeric mixture of conjugated linoleic acids but not pure cis-9,trans-11-octadecadienoic acid affects body weight gain and plasma lipids in hamsters. *J Nutr*, **130**: 27-9.

23. STANGL GI (2000). High dietary levels of a conjugated linoleic acid mixture alter hepatic glycerophospholipid class profile and cholesterol-carrying serum lipoproteins of rats. *J Nutr Biochem*, **11**: 184-91.

24. DE DECKERE EAM, VAN AMELSVOORT JMM, MCNEIL GP, JONES P (1999). Effects of conjugated linoleic acid (CLA) isomers on lipid levels and peroxisome proliferation in the hamster. *Br J Nutr*, **82**: 309-17.

25. MUNDAY JS, THOMPSON KG, JAMES KAC (1999). Dietary conjugated linoleic acids promote fatty streak formation in the C57BL/6 mouse atherosclerosis model. *Br J Nutr*, **81**: 251-5.

26. LEE KN, KRITCHEVSKY D, PARIZA MW (1994). Conjugated linoleic acid and atherosclerosis in rabbits. *Atherosclerosis*, **108**: 19-25.

27. KRITCHEVSKY D, TEPPER SA, WRIGHT S, TSO P, CZARNECKI SK (2000). Influence of conjugated linoleic acid (CLA) on establishment and progression of atherosclerosis in rabbits. *J Am Coll Nutr*, **19**: S472-7.

28. STANGL GI, MÜLLER H, KIRCHGESSNER M (1999). Conjugated linoleic acid effects on circulating hormones, metabolites and lipoproteins, and its proportion in fasting serum and erythrocyte membranes of swine. *Eur J Nutr*, **38**: 271-7.

29. COOK ME, MILLER CC, PARK Y, PARIZA M (1993). Immune modulation by altered nutrient metabolism: nutritional control of immune-induced growth depression. *Poult Sci*, **72**: 1301-5.

30. PARK Y (1996). *Regulation of energy metabolism and the catabolic effects of immune stimulation by conjugated linoleic acid*. PhD dissertation. University of Wisconsin.

31. DE VONEY D, PARIZA M, COOK HW (1997). Conjugated linoleic acid increases blood and splenic T-cell response post lipopolysaccharide injection. *FASEB J*, **9**: 3355.

32. BERVEN G, BYE A, HALS O, et al. (2000). Safety of conjugated linoleic acid (CLA) in overweight or obese human volunteers. *Eur J Lipid Sci Technol*, **102**: 455-62.

33. GUDMUNDSEN O, WADSTEIN J, THOM E (2001). Human studies with CLA in Norway. In: *1st international conference on conjugated linoleic acid (CLA)*, Alesund (Norway).
34. VESSBY B, RISÉRUS U, SMEDANA A, BASU S (2001). Metabolic effects of conjugated linoleic acid (CLA) in humans. In: *1st international conference on conjugated linoleic acid (CLA)*, Alesund (Norway).

35. ZAMBELL KL, KEIM NL, VAN LOAN MD, *et al*. (2000). Conjugated linoleic acid supplementation in humans: effects on body composition and energy expenditure. *Lipids*, 35: 777-82.

36. MEDINA EA, HORN WF, KEIM NL, *et al*. (2000). Conjugated linoleic acid supplementation in humans: effects on circulating leptin concentrations and appetite. *Lipids*, 35: 783-8.

37. BENITO P, NELSON GJ, KELLEY DS, BARTOLINI G, SCHMIDT PC, SIMON V (2001). The effect of conjugated linoleic acid on platelet function, platelet fatty acid composition, and blood coagulation in humans. *Lipids*, 36: 221-7.

38. BOUGNOUX P, LAVILLONNIÈRE F, GARAUD P, JOURDAN ML, SÉBÉDIO JL, CHAJÈS V (2001). CLA in experimental mammary tumors and in breast cancer. In: *1st international conference on conjugated linoleic acid (CLA)*, Alesund (Norway).

39. KELLEY D, TAYLOR P, RUDOLPH I, *et al*. (2000). Dietary conjugated linoleic acid did not alter immune status in young healthy woman. *Lipids* 35: 1065-71.

40. MOHEDE I, ALBERS R, VAN DER WIELEN R, BRINK L, DOROVSKA-TARAN V (2001). Immuno-modulation: CLA stimulated antigen specific antibody production in humans. In: *1st international conference on conjugated linoleic acid (CLA)*, Alesund (Norway).

41. SCIMECA JA (1998). Toxicological evaluation of dietary conjugated linoleic acid in male Fischer 344 rats. *Fd Chem Toxic*, 36: 391-5.

42. YAMASAKI M, MANSHO K, MISHIMA H, *et al*. (1999). Dietary effect of conjugated linoleic acid on lipid levels in white adipose tissue of Spague-Dawley rats. *Biosci Biotechnol Biochem*, 63: 1104-6.

43. YAMASAKI M, MANSHO K, MISHIMA H, *et al*. (2000). Effects of dietary conjugated linoleic acid on lipid peroxidation and histological change in rat liver tissues. *J Agric Food Chem*, 48: 6367-71.

44. SUGANO M, TSUJITA A, YAMASAKI K, IKEDA I, KRITCHEVSKY D (1997). Lymphatic recovery, tissue distribution, and metabolic effects of conjugated linoleic acid in rats. *J Nutr Biochem*, 8: 38-43.

45. SUGANO M, TSUJITA A, YAMASAKI M, NOGUCHI M, YAMADA K (1998). Conjugated linoleic acid modulates tissue levels of chemical mediators and immunoglobulins in rats. *Lipids*, 33: 521-7.

46. MOYA-CAMARENA SY, VANDEN HEUVEL JP, BELURY MA (1999). Conjugated linoleic acid activates peroxisome proliferator-activated receptor alpha and beta subtypes but does not induce hepatic peroxisome proliferation in Sprague-dawley rats. *Biochim Biophys Acta*, 1436: 331-2.

47. YAMASAKI M, MANSHO K, MISHIMA H, *et al*. (2000). Effects of dietary conjugated linoleic acid on lipid peroxidation and histological change in rat liver tissues. *J Agric Food Chem*, 48: 6367-71.
48. BELURY MA, KEMPA-STECZKO A (1997). Conjugated linoleic acid modulates hepatic lipid composition in mice. *Lipids*, **32**: 199-204.

49. BELURY MA, MOYACAMARENA SY, LIU KL, HEUVEL JPV (1997). Dietary conjugated linoleic acid induces peroxisome-specific enzyme accumulation and ornithine decarboxylase activity in mouse liver. *J Nutr Biochem*, **8**: 579-84.

50. JONES PA, LEA LJ, PENDLINGTON RU (1999). Investigation of the potential of conjugated linoleic acid (CLA) to cause peroxisome proliferation in rats. *Fd Chem Toxic*, **37**: 1119-25.

51. BASU S, SMEDMAN A, VESSBY B (2000). Conjugated linoleic acid induces lipid peroxidation in humans. *FEBS Letters*, **468**: 33-6.

52. BASU S, RISÉRUS U, TURPEINEN A, VESSBY B (2000). Conjugated linoleic acid induces lipid peroxidation in men with abdominal obesity. *Clinical Science*, **99**: 511-6.

53. CHEW BP, WONG TS, SHULTZ TD, MAGNUSON NS (1997). Effects of conjugated dienoic derivatives of linoleic acid and beta-carotene in modulating lymphocyte and macrophage function. *Anticancer Res*, **17**: 1099-106.

54. HAYEK MG, HAN SN, WU DY, et al. (1999). Dietary conjugated linoleic acid influences the immune response of young and old C57BL/6NCrlBR mice. *J Nutr*, **129**: 32-8.

<figure><img src="image_1.png" /><figcaption>Figure. Urinary levels of 8-iso-PGF2alpha and 15-oxo-dihydro-PGF2alpha in control subjects and subjects treated with CLA for 1 month (from Basu et al. [52]).</figcaption></figure>
<table><caption>Table. Bioassay of mammary cancer prevention in rats fed CLA (adapted from Ip et al. [12]).</caption><thead><tr><th>Group</th><th>CLA in diet (g.L<sup>-1</sup>)</th><th>CLA composition</th><th>Tumor incidence</th><th>Total tumors (n)</th></tr></thead><tbody><tr><td>Control</td><td>0.1</td><td>9c,11t- 83%<br>7t,9c- 6%</td><td>28/30</td><td>92</td></tr><tr><td>Butter CLA</td><td>0.8</td><td>9c,11t- 92%<br>7t,9c- 5%</td><td>15/30</td><td>43</td></tr><tr><td>CLA mixture</td><td>0.8</td><td>9c,11t- 25%<br>10t,12c- 37%<br>11c,13t- 18%<br>8t,10c- 15%</td><td>17/30</td><td>48</td></tr></tbody></table>