Identification of the Conjugated Linoleic Acid Isomer Responsible for Inhibiting Essential Fatty Acid Metabolism and Tumor Cell Growth

David Won Lung Ma, Ph.D.

Thesis Abstract

Conjugated linoleic acid (CLA) refers to a group of geometrical and positional isomers of linoleic acid (LA) that have been shown to have beneficial health effects. Modulation of essential fatty acid metabolism by CLA may be a mechanism by which CLA inhibits tumorigenesis, however, the role of specific isomers is not known. Specific CLA isomers and their metabolites may compete and interfere with LA metabolism to AA, and reduce the synthesis of tumor promoting eicosanoids. Yet to be identified eicosanoids from CLA may also play a role in tumor growth.

Mechanistic studies in this area have been limited by methodological challenges. Availability of CLA from biological sources such as animal fat is low. Composition of commercial sources of CLA is variable and pure or enriched isomers are not available in quantities required for biological studies. Objectives of this study were (1) to develop a method to readily synthesize CLA from LA purified from safflower oil, (2) to separate and identify isomers by gas liquid chromatography, (3) to determine the level of CLA in Canadian beef and dairy foods, and (4) to develop a method to purify the major CLA isomers. These methods provide a foundation to study the effect of specific CLA isomers on essential fatty acid metabolism and eicosanoid synthesis.

Synthetic yields from safflower oil of upwards of 50% (wt/wt) CLA can be obtained, containing 45, 46.1 and 3.5% of the isomers, D9c,11t-, D10t,12c- and D9t,11t-18:2, respectively. Dairy and beef products contain the D9c,11t-18:2 isomer in a range between 1.2-6.2 mg/g fat or 0.03-81.0 mg/usual serving. Enriched mixtures of D9c,11t-, and D10t,12c-18:2 were prepared by urea crystallization. MDA-MB-231 mammary tumor cells supplemented with an equal mixture of D9c,11t- and D10t,12c-18:2, or mixtures enriched in either D9c,11t- or D10t,12c-18:2 were observed to preferentially incorporate D9c,11t-18:2. Among these three CLA treatments, membrane phospholipid levels of LA tended to increase and arachidonic acid (AA) was reduced specifically by the D10t,12c-18:2 isomer. The D10t,12c-18:2 isomer inhibited conversion of LA to AA. This corresponded with reduced PGE2 production and cell growth.

In summary, important methods of CLA analysis and preparation of enriched mixtures of CLA were developed. A greater understanding of the mechanism by which CLA inhibits tumor growth was also elucidated.