Opportunities for Basic and Clinical Research

on Conjugated Linoleic Acid

Submitted by

Clement Ip, Ph.D.

Dept. of Cancer Prevention

Roswell Park Cancer Institute

Buffalo, NY 14263

A conference, entitled “Perspectives on Conjugated Linoleic Acid Research: Current Status and Future Directions,” was held at the National Institutes of Health in May 15-16, 2002. The program content, abstracts, and some of the presentations can be found at http://ods.od.nih.gov/News/CLA_Conference.aspx. Conjugated linoleic acid (abbreviated to CLA) is a term which refers to a family of 18-carbon fatty acid isomers with two conjugated double bonds. Different CLA isomers have been identified based on the position as well as the arrangement of the double bonds. Along the acyl chain, the two conjugated double bonds could be in positions 7 and 9, 8 and 10, 9 and 11, 10 and 12, or 11 and 13. These are known as positional isomers. Additionally, there could be four possible spatial orientations to the two double bonds as denoted by the nomenclature of cis,cis (c,c); cis,trans (c,t); trans,cis (t,c) and trans,trans (t,t). These are known as geometric isomers. Sophisticated analytical techniques have only recently been developed to separate the spectrum of CLA isomers.

Dairy products are rich sources of CLA, in particular the c9,t11-CLA isomer. The microbial biohydrogenation of linoleic acid to stearic acid involves the formation of c9,t11-CLA as the first step, catalyzed by an isomerase-mediated reaction unique to bacteria in the rumen of dairy cows. A portion of CLA in milk fat arises from CLA that has escaped complete rumen biohydrogenation. A more significant portion of CLA in dairy cows is synthesized in the tissues by D9-desaturase from vaccenic acid (t11-18:1), another intermediate in the rumen biohydrogenation pathway. Over 90% of CLA in food is the c9,t11-CLA isomer. CLA can also be made synthetically from linoleic acid by the process of heat alkaline isomerization. Such a synthetic preparation contains a mixture of CLA isomers. The two major ones are c9,t11-CLA and t10,c12-CLA; they constitute about 80% of the total in approximately 1:1 ratio. The remaining isomers are c7,t9-CLA, t8,c10-CLA and c11,t13-CLA. Most of the studies on CLA have used the synthetic mixed isomer preparation which is available commercially from a number of lipid specialty vendors. In the last few years, biological research with individual CLA isomers has focused exclusively on c9,t11-CLA and t10,c12-CLA. The reason for this is that highly purified preparations of these two CLA isomers have been successfully produced by various methodologies, while the other isomers have yet to be acquired in sufficient quantities. In order to simplify the complicated terminologies, c9,t11-CLA is abbreviated to 9,11-CLA, and t10,c12-CLA to 10,12-CLA.

Numerous physiological properties have been attributed to CLA. Health benefits include the action of CLA as an anti-carcinogenic, anti-atherosclerotic, anti-lipogenic and anti-diabetogenic agent. Furthermore, CLA is known to modulate lipid metabolism and immune function. A major focus of the conference was to review the literature on these topics. Once purified 9,11-CLA and 10,12-CLA became available, there was increasing awareness that specific CLA isomers may be responsible for certain biological or biochemical changes. CLA in the form of dietary supplements is available from health food stores in North America and Europe. Although some of these formulations are of dubious quality, for the most part they contain about 60% total of 9,11-CLA and 10,12-CLA as the major ingredients. Because of easy public access to CLA and the relative lack of oversight of label claims for dietary supplements, NIH is taking a closer look at the current status of CLA research.

The purpose of this report is not to recapitulate individual presentations. Instead, it is to identify research needs that will be helpful in advancing our knowledge of CLA nutrition and biology. From a human nutrition standpoint, the current food CLA database is inadequate to allow an assessment of CLA intake in a meaningful way. Since 9,11-CLA can be synthesized endogenously from vaccenic acid which is present in dairy products, the role of this biochemical reaction in contributing to tissue CLA accumulation needs to be evaluated systematically. An equally important issue is the development of biomarkers of chronic CLA intake. The above information is essential if we are to address the question of whether dietary sources alone (i.e. not as a supplement) are sufficient to realize the biological benefits of CLA.

CLA has been reported to cause body fat reduction in growing animals (rodents and pigs), and to depress milk fat synthesis in lactating cows and women. This effect appears to be unique to the 10,12-CLA isomer. In human studies which have used a mixture of 9,11-CLA and 10,12-CLA, the response of body fat reduction is much less consistent. The questions that remain unanswered are related to: (a) whether the impact of CLA on lipid metabolism is different in a growing animal versus a mature adult animal; (b) whether 9,11-CLA is able to modulate the effect of 10,12-CLA on lipid metabolism in certain cases; (c) whether the action of 10,12-CLA is primarily due to a suppression of de novo lipid synthesis or an increase of lipolysis; and (d) whether the contribution of these biochemical pathways may shift under various physiological conditions.

Although a number of epidemiological studies have examined the relationship between dairy product consumption and cancer risk, there are only two case-control studies which were designed specifically to investigate dietary/tissue CLA and cancer morbidity in humans. Both of these were targeted to breast cancer. Since these were dietary studies, the reference CLA is the 9,11-CLA isomer. The study from Finland reported that in postmenopausal women, dietary CLA and serum CLA were significantly lower in cases than in controls. The odds ratio, after adjustment for known risk factors of breast cancer, was 0.4 in the highest quintile versus the lowest, suggesting that CLA may have a protective effect against breast cancer. The second study was based in France, and breast fat CLA was used as an indicator of exposure. No protective effect of CLA on breast cancer risk was found. In view of the paucity of epidemiological studies on CLA and cancer, additional information in this area would be desirable.

CLA exerts a powerful anticancer effect in the rat mammary gland. There could be a number of underlying reasons to explain why CLA is particularly effective in mammary cancer prevention, at least in the animal model. The mammary epithelium is embedded in an adipocyte-rich stroma, and importantly, CLA is incorporated preferentially into adipocyte neutral lipid. CLA released from mammary adipocytes may serve as a paracrine factor to block the clonal expansion of premalignant epithelial cells and the differentiation of stromal-vascular stem cells to endothelial cells. Inhibition of angiogenesis may represent a novel mechanism in the anti-cancer effect of CLA. This could be a potentially fruitful area of research in the future.

Although CLA has been shown to protect against tumorigenesis in other animal models, the database is far less extensive, and in most cases, the experiments have not been replicated sufficiently to reach consensus. Furthermore, there is a vacuum of knowledge regarding the activity of CLA isomers in animal carcinogenesis models. Cell culture studies have been used to investigate the effect of CLA on proliferation and apoptosis. However, the physiological relevance of these in vitro findings is questionable since many of these experiments were done with very high levels of CLA dissolved in an organic solvent.

The anti-diabetogenic effect of CLA is interesting but is very poorly defined; there is little information in the literature on this subject. The presence of confounding factors associated with the experimental model, the Zucker diabetic fatty rat further contributes to our lack of a clear understanding of the ability of CLA to lower blood glucose. These factors include rodent strain differences, the response of pre-diabetic versus diabetic conditions, insulin resistance effect, reduced body fat mass, changing blood lipid profile, as well as the duration and dose of CLA treatment. These issues need to be resolved before undertaking any controlled intervention trials in humans.

Research on the biology of CLA must be accompanied by equal attention to two fundamental aspects of CLA biochemistry. It has been well documented that CLA is metabolized to downstream elongation and desaturation products following the pathway of linoleic acid metabolism. The identification of conjugated arachidonic acid in particular has engendered a great deal of interest because of the potential that it may: (a) deplete the availability of arachidonic acid; (b) interfere with cyclooxygenase- or lipoxygenase-mediated biosynthesis of eicosanoids from arachidonic acid; or (c) produce its own eicosanoid analogs which may have unique biological activities. Thus the role of CLA metabolism in contributing to the effect of CLA should be an area of future research emphasis. A second area that is begging for attention is related to the intracellular signaling mechanism of CLA or CLA metabolites. CLA has been shown to be a high affinity ligand and activator of peroxisome proliferator-activated receptors (PPAR), a family of transcription factors known to affect gene expression in a tissue-specific manner. Isoforms of PPAR are widely distributed, although in highly variable amounts, in different tissues. Comprehensive studies are needed to elucidate the significance of individual PPAR family members in mediating the effects of CLA in a given organ. In this regard, the use of cutting-edge genomics technology would be a valuable tool to characterize signaling pathways and molecular targets that are relevant to the action of CLA under defined conditions. Other candidates for mediating the transcriptional effects of CLA may include the sterol-regulatory binding protein (SREBP) family, the activity or expression of which has been shown to be altered by polyunsaturated fatty acids, but for which the effects of CLA have not yet been investigated. Finally, the many biological activities attributed to individual CLA isomers suggest that there is a high degree of tissue specificity with respect to the mechanism by which CLA and/or its metabolites exert their effects. Unraveling this complexity at the cellular and molecular levels will be the challenge of the CLA field in the years to come.