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Rakesh Kapoor, Martin Reaney, and Neil D. Westcott
Bioriginal Food and Science Corp. Saskatoon, Saskatchewan, Canada
The discovery of conjugated linoleic acid (CLA) dates back to 1933, when it was found that treatment of polyunsaturated fatty acids with alkali increased the UV absorbency. It was later found that the treatment produced a one to one mixture of cis-9-, trans-11- (9,11-ct) and trans-10-, cis-12 (10,12-tc) CLA. In 1935, it was noted that UV absorbency at 230 nm of milkfats was higher in milk from cows fed polyunsaturated fatty acids than cows fed saturated fats. This phenomenon was shown to be a result of conjugation of the double bonds of the polyunsaturated fatty acids. The predominant isomer in dairy products (milk, cheese, butter) or meat is 9,11-ct CLA.
Since the time of their discovery, conjugated fatty acids have been the subject of intense investigation. Spectroscopic analysis using ultraviolet light was the major analytical instrument available to researchers in the 1930s. As a result of high absorbance of UV light at 230 nm or higher, conjugated fat became a useful research tool for the study of fat metabolism. The first animalstudy used naturally conjugated fats of tung oil, but it was poorly tolerated. During this period, essential fatty acids were discovered. Aaes-Jorgensen studied the possibility of using CLA to treat/prevent the symptoms of essential fatty acid-deficiency. They observed that in essential fatty acid-deficient animals, CLA could not prevent deficiency but showed toxicity. Synthetic conjugated fatty acids produced from linoleic acid (LA), usually mixtures of 9,11-ct and 10,12-tc CLA, replaced the natural substances as a preferred biological marker.
Over the last two decades, the conjugated fats and, particularly, conjugated linoleic acid (CLA) have been intensively studied for their biological activity. As a result of the ease of synthesis, blends of two CLA isomers, namely 9,11-ct and 10,12-tc-CLA, have been the focus of most research into biological activity. Recent research, however, has been expanding to include pure or enriched isomer preparations.
Early studies using a CLA mixture revealed that animals absorb and incorporate some of the CLA in their tissues in phospholipid, glycolipid, and acylglycerol fractions. In 1950, Reiser observed faster and better absorption and incorporation of CLA when administered as triacylglycerol (TAG) versus free fatty acid. Following administration of CLA as TAG, the maximal levels appear in blood, liver, and organs at 16 hours compared with 24 hours for the free fatty acid form. Incorporation was higher in mesentery fat followed by perirenal and subcutaneous fat. Barnes et al., in 1941, reported the kinetics of CLA absorption from the mixture. The absorption rate was highest in the first hour following administration and then gradually declined. Following a single oral dose, over 50% of conjugated CLA was absorbed in neutral mucosal lipids in the first hour followed by a decline, whereas in mucosal phospholipids, the incorporation was much slower, reaching maximum at about 8 hours followed by decline. In 1951, it was found that poultry incorporate CLA into egg lipids. Recent studies confirm the earlier finding of preferential incorporation in neutral lipids followed by phospholipids in liver and mammary tissues. Incorporation of CLA in tissues is associated with a reduction in the amounts of arachidonic acid and linoleic acid in neutral lipids and in the liver TAG levels with an increase in the levels of 18:0. In pig heart lipids, 11,13-ct isomer was the major CLA isomer followed by 9,11-ct isomer following an oral administration of CLA mixture. CLA isomers compete with desaturases and elongases to produce desaturated and elongated products maintaining the geometry of double bonds. CLA has been shown to inhibit delta-9-desaturase enzyme in vitro and in vivo. CLA feeding also resulted in a decrease in arachidonic acid, and also reduced the desaturation of linoleic acid without affecting the desaturation of alpha-linolenic acid to any significant level. In rat, 9,11-ct isomer was preferentially metabolized to conjugated C20:3, whereas 10,12-tc isomer was metabolized to conjugated C16:2 and 18:3 compounds. The CLA mixture and its individual isomers (9,11-ct and 10,12-tc) inhibited basal and calcium ionophore stimulated production of prostaglandin from human sapheneous vein endothelial cells in a dose-dependent manner. The mixture of CLA isomers was reported to inhibit the production of eicosanoids at all doses, whereas 10,12-tc isomer was shown to inhibit production at lower dose but stimulate at higher dose.
3. PHYSIOLOGICAL ACTIONS OF CLA
3.1. Effect on Body Composition
While working with essential fatty acid deficient rats in 1951, Holman observed that CLA-fed rats had significantly less total fat than control rats, and that they lost weight. Subsequent studies demonstrated that CLA inhibited fat accumulation and promoted lean muscle mass in growing animals, including pigs and mice. The results on the effect of CLA on body fat composition in animals are unequivocal, whereas studies in humans are providing mixed results. In a double blind, randomized clinical trial on obese and overweight humans, Blankson et al. observed a clinically significant reduction in body fat mass in groups administered various doses of CLA ranging from 1.7 g per day to 6.8 g per day. Reduction in body fat mass was significant for groups administered 3.4 g CLA per day and 6.8 g CLA per day. Interestingly, this study found that 3.4 g of CLA per day provided maximum reduction in body fat mass; increasing the dose above this level provided no additional effect. Lean body mass and body mass index were similar in all the groups, although there was a slight increase in lean body mass in the CLA group. The increase in lean body mass did not achieve significance compared with a placebo group administered olive oil. Additionally, the CLA group presented a significant reduction in total-, LDL-, and HDL-cholesterol. In another study, Riserus et al. observed a reduction in saggital abdominal diameter in abdominally obese humans without affecting total body weight. A reduction in total fat mass in healthy, nonobese, exercising males was observed when CLA was given at a total daily dose of 1.8 g (divided in 3 doses) for 12 weeks. Body weight was not affected in this double blind clinical trial. A recent study in type II diabetic patients who were not on any medication found an inverse relationship between plasma CLA levels and weight loss and serum leptin levels. The inverse relationship was significant for 10,12-tc isomer of CLA and not for 9,11-ct isomer. A study in nonobese individuals using a mixture of CLA isomers containing about 20% each of 9,11-ct and 10,12-tc-CLA isomers, along with 20% to 25% other isomers, did not observe a reduction in body fat mass. Another study investigated the effect of CLA on weight regain after weight loss in overweight subjects. This study observed no effect of CLA on weight gain after weight loss; however, the weight gain in the CLA group was a result of an increase in fat free mass and was independent of dose. Comparison between studies is difficult as these studies differed in the degree of obesity of the subjects, duration of treatment, dose, and the isomer composition of the CLA preparation. Earlier commercial products of CLA contained equal amounts of 9,11-ct and 10,12 -tc isomers with other isomers in small amounts. The other isomers include all trans-isomers as well as other cis-, trans-isomers, including 11,13-ct, 11,13-tc, 8,10 ct,8,10-tc, etc. This illustrates the need for research on specific isomers and standardized protocols.
These reported actions of CLA could be mediated through a number of physiological mechanisms including increased fat oxidation or inhibition of lipid accumulation in fat cells. Recent studies have started to investigate the physiological actions of individual isomers. It appears that the 10,12-tc isomer of CLA is mainly responsible for the effect of CLA on adiposity.
It was demonstrated that 10,12-tc-CLA reduces leptin, a hormone involved in regulation of fat deposition, in cultured fat cells, and in mice. In the latter study, feeding 10,12-tc-CLA to mice caused a comparatively small gain in weight with no gain in adipose fat. The 10,12-tc isomer of CLA was also shown to inhibit differentiation of preadipocytes in murine (3T3-L1) and human preadipocytes. This was associated with decreased accumulation of TAGs in differentiating preadipocytes; inhibition of peroxisome proliferator-activated receptor -[gamma] (PPAR-[gamma]) gene and its downstream gene products including lipoprotein lipase (LPL), GLUT-4 (glucose transporter gene 4), and inhibited expression of fatty acid synthase (FAS) gene. CLA isomer 9,11-ct, on the other hand, increased accumulation of TAGs in adiposities and also stimulated GLUT-4 and LPL. This suggests that isomer 10,12-tc may be responsible for inhibition of glucose uptake and oxidation in the adipocytes, leading to decreased TAG accumulation. These actions also underlie the effect of 10,12-tc isomer in inducing insulin resistance leading to lipoatrophic diabetes observed in animal and human studies.
3.2. Anticancer Properties
In 1985, Pariza and Hargrave discovered an antimutagenic fraction in cooked and raw beef during their studies on identification of carcinogenic compounds present in cooked beef. This fraction was identified to be a mixture of 4 isomers of CLA (9,11-ct, 9,11-tt, 10,12-tc, and 10,12-tt). Studies in animal models and cell lines demonstrated the antimutagenic activity of a CLA mixture against known chemical carcinogens (7,12-dimethylbenz[a]anthracene, DMBA, and benzo (a) pyrene). CLA has been shown to have anticancer effects against breast, colon, and prostate cancer cell lines. In rat models of breast cancer, CLA was reported to affect the breast structure when given during development stages. In this study, dietary CLA reduced the proliferation of terminal end bud and lobuloalveolar bud structures, whereby breast tissue became resistant to neoplastic transformations associated with cancer at later stages in life.
The exact mechanism of anticancer effects of CLA is not clear, and several possible mechanisms could underlie the anticancer properties of CLA. These actions may include its ability to interfere with the proliferation of cancer cells, increased apoptotic cell death, inhibition of angiogenesis, or increased oxidative stress. In a study comparing the effects of CLA on estrogen receptor positive human breast cancer cells (MCF-7) and estrogen receptor negative (MDA-MB 231) cells, CLA was shown to selectively inhibit proliferation of estrogen receptor positive cells. CLA-treated MCF-7 cells selectively remained in G0/G1 phase and the expression of c-myc was inhibited. CLA had no effect on the growth of MDA-MB 231 cells. This study suggests that CLA acts by interfering with the estrogen-mediated second messenger system. CLA is also known to interfere with eicosanoids pathway and inhibits production of prostaglandin [E.sub.2] (PG[E.sub.2]). Reduced production of PGE2 may play a role in anticancer actions of CLA. CLA is also shown to stimulate apoptotic death of cancer cells. CLA isomers increased apoptosis by stimulating the expression of caspase 3 and 9 activities and by reducing the expression of Bcl-2, an apoptosis repressor gene. The 10,12-tc isomer of CLA was found to be more potent in mediating these actions than either the 9,11-ct isomer or a mixture of the two. The other possible mechanism for anticancer properties of CLA is its ability to inhibit angiogenesis. In a mouse model of breast cancer, both isomers inhibited angiogenesis in mammary fat pad and reduced the concentration of vascular endothelium-derived growth factor. The CLA isomer 10, 12-tc also inhibited secretion of leptin and induced apoptosis in white and brown adipocytes, whereas 9,11-ct isomer was without effect on these parameters. CLA was also shown to reduce cell proliferation by reducing the expression of proteins involved in cell cycle regulation (p16 and p27) and DNA synthesis.
The above discussion focused on the role of CLA as a chemoprotective agent. Information regarding its effect on cancer treatment is limited. Feeding CLA for four or eight weeks after carcinogen administration was reported to be ineffective in preventing tumor formation, whereas continuous administration protected against tumor development. A recent case control study in Finnish women suggested that dietary CLA may be protective against breast cancer. The role of CLA in prevention or treatment of cancer in humans is not clear and requires more research.
3.3. Insulin Resistance and Diabetes
CLA has been shown to normalize impaired glucose tolerance and improve hyper-insulinemia in prediabetic ZDF rats. These actions appeared to be mediated through PPAR-[gamma] pathway as CLA treatment induced expression of mRNA for aP2. Recently, it has been observed in Zucker diabetic rats that either a 50:50 mixture of 9,11-ct-CLA and 10,12-tc-CLA isomers or a 90% 9,11-ct-CLA isomer stimulated insulin action in fat and muscle cells. These observations suggest that CLA can prevent or delay the onset of diabetes and that the 9,11-ct isomer may contribute much of this activity. Recently, 10,12-tc isomer of CLA was shown to induce insulin resistance and hyperinsulinemia in mice, whereas 9,11-ct isomer had no effect. Both these isomers were shown to be equally efficient in stimulating PPAR-[alpha] and [gamma] receptors, indicating that the hyperinsulinemia may not be mediated through nuclear receptor pathway. In another study using either high metabolic rate mice or low metabolic rate mice, Hargrave et al. demonstrated that CLA increased the insulin resistance in high metabolic rate mice only, whereas in Zucker diabetic rat, CLA was shown to prevent a rise in insulin and glucose levels that might have been mediated through an increased production of adiponectin, a hormone released by adipose tissues. In pigs, dietary CLA had no effect on plasma glucose or insulin levels or on the ability of insulin to mobilize plasma glucose. These studies indicate that the effect of CLA in diabetes is not clear and the reported differences may be species specific. In human subjects, the effects of CLA on insulin resistance and glucose homeostasis are not well studied. In one clinical trial, the 10,12-tc isomer, but not a mixture of 9,11-ct and 10,12-tc isomers, was shown to increase the insulin resistance and blood glucose levels in abdominally obese people, whereas another study on normolipidemic subjects failed to observe any effect of either a 50:50 or 80:20 mixture of 9,11-ct and 10,12-tc isomers on blood glucose or insulin levels. Belury et al. observed a reduction in fasting plasma glucose levels in type II diabetics when they were treated with 6.0 g of a mixture of CLA for eight weeks. This necessitates the need for controlled studies in human to delineate the effect of CLA and its isomers on blood glucose homeostasis and insulin resistance.
3.4. Cardiovascular Actions
Excerpted from Bailey's Industrial Oil and Fat Products, Edible Oil and Fat Products Copyright © 2005 by John Wiley & Sons, Inc.. Excerpted by permission.
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