Conjugated Linoleic Acid (CLA)

Antiplatelet effects of conjugated linoleic acid isomers.


Truitt A; McNeill G; Vanderhoek JY. Department of Biochemistry and Molecular Biology, The George Washington University, 2300 Eye Street, N.W., Washington, DC 20037, USA Biochim Biophys Acta 1999 May 18; 1438(2): 239-246 PMID: 10320806 UI: No Cit. ID assigned

Conjugated diene isomers of linoleic acid (CLA) are normal constituents of certain foods and exhibit anticarcinogenic and antiatherogenic properties. In the present study, the effects of several CLA isomers on human platelet aggregation and arachidonic acid metabolism were examined. It was found that 9c,11t-CLA, 10t,12c-CLA and 13-hydroxy- 9c,11t-octadecadienoic acid (13-HODE) inhibited arachidonic acid- and collagen-induced platelet aggregation with I50s in the 5-7 #M range. The nonconjugated 9c,12c-LA was about 300% and 50%, respectively, less potent an inhibitor with these aggregating agents. Using either thrombin or the calcium ionophore A23187 as aggregating agents, a CLA isomer mix was also found to be more inhibitory than 9c,12c-LA. The 9c,11t- and 10t,12c-CLA isomers as well as the CLA isomer mix inhibited formation of the proaggregatory cyclooxygenase-catalyzed product TXA2, as measured by decreased production of its inactive metabolite [14C]TXB2 from exogenously added [14C]arachidonic acid (I50s=9-16 #M). None of the CLA isomers tested inhibited production of the platelet lipoxygenase metabolite [14C]12-HETE. The additional presence of a hydroxyl group gave opposite results: 13-HODE (I50=3 #M) was about 4- fold more potent a cyclooxygenase inhibitor than the 9c,11t-CLA isomer but 9-HODE was 2- to 3-fold less effective an inhibitor (I50=34 #M) of [14C]TXB2 formation than the corresponding 10t,12c-CLA. In both the aggregation and arachidonic acid metabolism experiments, the inhibitory effects of CLA on platelets were reversible and dependent on the time of addition of either the aggregating agent or the [14C] arachidonic acid substrate. These studies suggest that CLA isomers may also possess antithrombotic properties.


Changes in body composition in mice during feeding and withdrawal of conjugated linoleic acid

Park Y; Albright KJ; Storkson JM; Liu W; Cook ME; Pariza MW Food Research Institute, Department of Food Microbiology and Toxicology, University of Wisconsin- Madison 53706, USA. Lipids 1999 Mar;34(3):243-8 PMID: 10230717 UI: 99245748

Two experiments were conducted. In Experiment 1, 8-wk-old mice were fed control diet or diet supplemented with 0.5% conjugated linoleic acid (CLA) to study the effect of CLA on body composition (CLA: 40.8-41.1% c- 9,t-11 isomer, 43.5-44.9% t-10,c-12 isomer). The data for CLA-fed mice vs. controls described parallel but significantly distinct responses for both absolute and relative changes in body fat mass (reduced in CLA- fed mice) and for relative changes in whole body protein and whole body water (both of which were increased in CLA-fed mice). In the CLA-fed mice, the effect on whole body protein appeared to precede the reduction in body fat mass. In Experiment 2, weanling mice were fed control diet or diet supplemented with 0.5% CLA for 4 wk (test group), at which time all mice were fed control diet devoid of added CLA. The test group exhibited significantly reduced body fat and significantly enhanced whole body water relative to controls at the time of diet change. Time trends for changes in relative body composition were described by parallel lines where the test group exhibited significantly less body fat but significantly more whole body protein, whole body water, and whole body ash than controls. Tissue CLA levels declined following the withdrawal of CLA from the diet. In skeletal muscle of mice fed CLA-supplemented diet, the t- 10,c-12 isomer was cleared significantly faster than the c- 9,t-11 CLA isomer.


Evidence that the trans-10,cis-12 isomer of conjugated linoleic acid induces body composition changes in mice

Park Y; Storkson JM; Albright KJ; Liu W; Pariza MW. Food Research Institute, Department of Food Microbiology and Toxicology, University of Wisconsin-Madison 53706, USA. Lipids 1999 Mar; 34(3): 235-41 PMID: 10230716 UI: 99245747

We investigated the effects of conjugated linoleic acid (CLA) preparations, which were enriched for the cis- 9,trans-11 CLA isomer or the trans-10,cis-12 CLA isomer, on body composition in mice. Body composition changes (reduced body fat, enhanced body water, enhanced body protein, and enhanced body ash) were associated with feeding the trans-10,cis-12 CLA isomer. In cultured 3T3-L1 adipocytes, the trans- 10,cis-12 isomer reduced lipoprotein lipase activity, intracellular triacylglycerol and glycerol, and enhanced glycerol release into the medium. By contrast, the cis-9,trans-11 and trans-9,trans- 11 CLA isomers did not affect these biochemical activities. We conclude that CLA-associated body composition change results from feeding the trans- 10,cis- 12 isomer.


An increase in vitamin a status by the feeding of conjugated linoleic acid

Banni S; Angioni E; Casu V; Melis MP; Scrugli S; Carta G; Corongiu FP; Ip C Dipartimento di Biologia Sperimentale, Universitale degli Studi di Cagliari, Cittadella Universitaria, Italy. Nutr Cancer 1999; 33(1): 53-7 PMID: 10227044 UI: 99243631

Previous research indicated that conjugated linoleic acid (CLA) is a potent inhibitor of mammary carcinogenesis. The present study showed a progressive increase in retinol (vitamin A alcohol) in the liver in proportion to CLA intake in rats that were fed different levels of CLA (in increments of 0.5%) for 1 month. The escalation reached a magnitude of about fivefold over the control at 2% dietary CLA. In contrast, the increase in liver retinyl esters peaked at about twofold between 0.5% and 1% CLA. Only retinol was detected in mammary tissue; a maximal twofold increase was attained at 0.5% CLA, and no dose-response effect was evident. The above findings are discussed in relation to two important questions: 1) How does CLA raise vitamin A status in the animal? 2) Is the increase in vitamin A associated with the anticarcinogenic effect of CLA?


Feeding conjugated linoleic acid to animals partially overcomes catabolic responses due to endotoxin injection.

Miller CC; Park Y; Pariza MW; Cook ME. Poultry Science Dept., U.W. Madison 53706. Biochem Biophys Res Commun 1994 Feb 15;198(3):1107-12 PMID: 8117267 UI: 94161721

The ability of conjugated linoleic acid to prevent endotoxin-induced growth suppression was examined. Mice fed a basal diet or diet with 0.5% fish oil lost twice as much body weight after endotoxin injection than mice fed conjugated linoleic acid. By 72 hours post injection, mice fed conjugated linoleic acid had body weights similar to vehicle injected controls; however, body weights of basal and fish oil fed mice injected with endotoxin were reduced. Conjugated linoleic acid prevented anorexia from endotoxin injection. Splenocyte blastogenesis was increased by conjugated linoleic acid.


Combined silver-ion and reversed-phase high-performance liquid chromatography for the separation and identification of C20 metabolites of conjugated linoleic acid isomers in rat liver lipids

Juaneda P; Sebedio JL. INRA, Unite de nutrition Lipidique, Dijon, France. Pierre.Juaneda@d ijon.inra.fr J Chromatogr B Biomed Sci Appl 1999 Mar 19;724(2):213-9 PMID: 10219661 UI: 99236401

Reversed-phase high-performance liquid chromatography (RP- HPLC) and silver-ion high-performance liquid chromatography (Ag-HPLC) were successively combined for the separation of the longer-chain metabolites of conjugated linoleic acids (CLAs). Commercial silver nitrate-impregnated columns were used with an eluting solvent composed of a mixture of hexane-acetonitrile. Fatty acid methyl esters (FAMEs) from liver lipids of rats fed CLA were analysed. This method allowed separation both of the non-conjugated FAME, as C16: 1, C18: 2, C18: 3, C20: 4 and C22: 5, but also the conjugated fatty acids like CLA, 8,12,14- 20:3, 5,8,12,14-20:4 and 5,8,11,13- 20:4. The presence of 8,11,13-20:3 is reported for the first time. This method is of interest for the isolation and identification of the C20 conjugated metabolites that cannot be resolved by gas chromatography. Furthermore, it allows the isolation of FAME for further characterisation by GC-mass spectrometry (MS).


Conjugated linoleic acid rapidly reduces body fat content in mice without affecting energy intake.

DeLany JP; Blohm F; Truett AA; Scimeca JA; West DB. Pennington Biomedical Research Center, Louisiana State University, Baton Rouge 70808, Louisiana, USA. delanyjp/@mhs.pbrc.edu Am J Physiol 1999 Apr; 276(4 Pt 2): R1172-9 PMID: 10198400 UI: 99216258

Recent reports have demonstrated that conjugated linoleic acid (CLA) has effects on body fat accumulation. In our previous work, CLA reduced body fat accumulation in mice fed either a high-fat or low-fat diet. Although CLA feeding reduced energy intake, the results suggested that some of the metabolic effects were not a consequence of the reduced food intake. We therefore undertook a study to determine a dose of CLA that would have effects on body composition without affecting energy intake. Five doses of CLA (0.0, 0.25, 0.50, 0.75, and 1.0% by weight) were studied in AKR/J male mice (n = 12/group; age, 39 days) maintained on a high-fat diet (%fat 45 kcal). Energy intake was not suppressed by any CLA dose. Body fat was significantly lower in the 0.50, 0.75, and 1.0% CLA groups compared with controls. The retroperitoneal depot was most sensitive to the effects of CLA, whereas the epididymal depot was relatively resistant. Higher doses of CLA also significantly increased carcass protein content. A time- course study of the effects of 1% CLA on body composition showed reductions in fat pad weights within 2 wk and continued throughout 12 wk of CLA feeding. In conclusion, CLA feeding produces a rapid, marked decrease in fat accumulation, and an increase in protein accumulation, at relatively low doses without any major effects on food intake.


LDL isolated from Greek subjects on a typical diet or from American subjects on an oleate-supplemented diet induces less monocyte chemotaxis and adhesion when exposed to oxidative stress.

Tsimikas S; Philis-Tsimikas A; Alexopoulos S; Sigari F; Lee C; Reaven PD. Division of Cardiovascular Diseases, Department of Medicine, University of California at San Diego, La

Jolla, California, USA. Arterioscler Thromb Vasc Biol 1999 Jan; 19(1): 122-30 PMID: 9888874 UI: 99106030

The mechanisms underlying the cardiovascular benefits of Mediterranean- style diets are not fully understood. The high content of monounsaturated fatty acids in Mediterranean-style diets derived from oleate-rich olive oil may be beneficial in reducing low density lipoprotein (LDL) oxidation and its subsequent development of atherogenic properties. This study sought to assess the proinflammatory potential of LDL isolated from subjects consuming a diet naturally rich in olive oil. LDL was isolated from 18 Greek, 18 American, and 11 Greek- Americans subjects, all of whom were living in the United States. Fatty acid composition and vitamin E levels of LDL were determined, as was the extent of copper-mediated LDL oxidation. LDL was also mildly oxidized by exposure to fibroblasts overexpressing 15-lipoxygenase and tested in vitro for bioactivity by determining its ability to stimulate monocyte chemotaxis and adhesion to endothelial cells. To confirm that dietary fatty acids influence the proinflammatory properties of mildly oxidized LDL, LDL was also isolated from 13 healthy American subjects after consumption of an 8-week liquid diet supplemented with either oleic (n=6) or linoleic (n=7) acid and tested for bioactivity in a similar fashion. There were no differences in the baseline lipid profiles among the Greeks, Americans, or Greek-Americans. Oleic acid content in LDL was 20% higher in the Greek compared with the American or Greek-American subjects (P<0.001). The extent of in vitro LDL oxidation, measured by conjugated diene formation, was lower in the Greek subjects (P<0.02), but there was no difference in the lag time. Induction of monocyte chemotaxis and adhesion by mildly oxidized LDL was decreased by 42% in the Greek group compared with the American subjects (P<0.001). There was an inverse correlation between the oleic acid content of LDL and stimulation of monocyte chemotaxis (r=-0.64, P<0.001) and a positive correlation between the polyunsaturated fatty acid content of LDL (total linoleate and arachidonic acids levels in LDL) and stimulation of monocyte chemotaxis (r=0.51, P<0.01) in the entire cohort. There were no differences in LDL vitamin E content between the groups. In the liquid-diet groups, the oleic acid- supplemented group had a 113% higher oleic acid content in LDL and a 46% lower linoleic acid content in LDL than the linoleate-supplemented group (P<0.001), whereas the vitamin E content in LDL was equal in both groups. When exposed to oxidative stress, the LDL enriched in oleic acid promoted less monocyte chemotaxis (52% lower) and reduced monocyte adhesion by 77% in comparison with linoleate-enriched LDL (P<0.001). There was a strong, negative correlation between oleic acid LDL content and monocyte adhesion (r=- 0.73, P<0.001) and a strong, positive correlation between polyunsaturated fatty acid LDL content and monocyte adhesion (r=0.87, P<0.001). This study demonstrates that dietary enrichment of LDL with oleic acid is realistic and readily achieved by using diets currently in use in Mediterranean countries. In addition, these data suggest that LDL enriched with oleic acid and reduced in polyunsaturated fatty acids may be less easily converted to a proinflammatory, minimally modified LDL.


Effects of conjugated linoleic acid on body fat and energy metabolism in the mouse.

West DB; Delany JP; Camet PM; Blohm F; Truett AA; Scimeca J. Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana 70808, USA. Am J Physiol 1998 Sep; 275(3 Pt 2): R667-72 PMID: 9728060 UI: 98399776


Conjugated linoleic acid (CLA) is a naturally occurring group of dienoic derivatives of linoleic acid found in the fat of beef and other ruminants. CLA is reported to have effects on both tumor development and body fat in animal models. To further characterize the metabolic effects of CLA, male AKR/J mice were fed a high-fat (45 kcal%) or low- fat (15 kcal%) diet with or without CLA (2.46 mg/kcal; 1.2 and 1.0% by weight in high- and low-fat diets, respectively) for 6 wk. CLA significantly reduced energy intake, growth rate, adipose depot weight, and carcass lipid and protein content independent of diet composition. Overall, the reduction of adipose depot weight ranged from 43 to 88%, with the retroperitoneal depot most sensitive to CLA. CLA significantly increased metabolic rate and decreased the nighttime respiratory quotient. These findings demonstrate that CLA reduces body fat by several mechanisms, including a reduced energy intake, increased metabolic rate, and a shift in the nocturnal fuel mix.


Opposite effects of linoleic acid and conjugated linoleic acid on human prostatic cancer in SCID mice.

Cesano A; Visonneau S; Scimeca JA; Kritchevsky D; Santoli D. Wistar Institute, Philadelphia, PA 19104, USA. Anticancer Res 1998 May-Jun; 18(3A): 1429-34 PMID: 9673351 UI: 98338068

The relationship between dietary fat intake (level and type) and cancer development is a matter of concern in Western society. The purpose of this study was to determine the effect of three different diets on the local growth and metastatic properties of DU-145 human prostatic carcinoma cells in severe combined immunodeficient (SCID) mice. Animals were fed a standard diet or diets supplemented with 1% LA or 1% CLA for 2 weeks prior to subcutaneous (s.c.) inoculation of DU-145 cells and throughout the study (total of 14 weeks). Mice receiving LA- supplemented diet displayed significantly higher body weight, lower food intake and increased local tumor load as compared to the other two groups of mice. Mice fed the CLA-supplemented diet displayed not only smaller local tumors than the regular diet-fed group, but also a drastic reduction in lung metastases. These results support the view that dietary polyunsaturated fatty acids may influence the prognosis of prostatic cancer patients, thus opening the possibility of new therapeutic options.


Conjugated linoleic acids alter bone fatty acid composition and reduce ex vivo prostaglandin E2 biosynthesis in rats fed n-6 or n-3 fatty acids.


Li Y; Watkins BA. Department of Food Science, Purdue University, West Lafayette, Indiana 47907, USA. Lipids 1998 Apr; 33(4): 417-25 PMID: 9590630 UI: 98250512

This study evaluated the effects of conjugated linoleic acids (CLA) on tissue fatty acid composition and ex vivo prostaglandin E2 (PGE2) production in rats given diets varying in n-6 and n-3 fatty acids. Four groups of rats were given a basal semipurified diet (AIN-93G) containing 70 g/kg of added fat for 42 d. The fat treatments were formulated to contain CLA (0 vs. 10 g/kg of diet) and n-6 (soybean oil having an n-6/n-3 ratio of 7.3) and n-3 fatty acids (menhaden oil + safflower oil having an n-6/n-3 ratio of 1.8) in different ratios in a 2 x 2 factorial design. Fatty acids in liver, serum, muscle, heart, brain, spleen, and bone (cortical, marrow, and periosteum) were analyzed by capillary gas-liquid chromatography. The various dietary lipid treatments did not affect growth; however, CLA improved feed efficiency. The CLA isomers were found in all rat tissues analyzed although their concentrations varied. Dietary CLA decreased the concentrations of 16:1n-7, 18:1, total monounsaturates and n-6 fatty acids, but increased the concentrations of n-3 fatty acids (22:5n-3 and 22:6n-3), and saturates in the tissues analyzed. Ex vivo PGE2 production in bone organ culture was decreased by n-3 fatty acids and CLA. We speculate that CLA reduced the concentration of 18:1 fatty acids by inhibiting liver delta9-desaturase activity. The fact that CLA lowered ex vivo PGE2 production in bone organ culture suggests that these conjugated fatty acids have the potential to influence bone formation and resorption.


Dietary conjugated linoleic acid normalizes impaired glucose tolerance in the Zucker diabetic fatty fa/fa rat [published erratum appears in Biochem Biophys Res Commun 1998 Jun 29; 247(3): 911]


Houseknecht KL; Vanden Heuvel JP; Moya-Camarena SY; Portocarrero CP; Peck LW; Nickel KP; Belury MA. Department of Animal Sciences, Purdue University, West Lafayette, Indiana 47907, USA. Biochem Biophys Res Commun 1998 Mar 27; 244(3): 678-82 PMID: 9535724 UI: 98205780

Conjugated linoleic acid (CLA) is a naturally occurring fatty acid which has anti-carcinogenic and anti- atherogenic properties. CLA activates PPAR alpha in liver, and shares functional similarities to ligands of PPAR gamma, the thiazolidinediones, which are potent insulin sensitizers. We provide the first evidence that CLA is able to normalize impaired glucose tolerance and improve hyperinsulinemia in the pre-diabetic ZDF rat. Additionally, dietary CLA increased steady state levels of aP2 mRNA in adipose tissue of fatty ZDF rats compared to controls, consistent with activation of PPAR gamma. The insulin sensitizing effects of CLA are due, at least in part, to activation of PPAR gamma since increasing levels of CLA induced a dose-dependent transactivation of PPAR gamma in CV-1 cells cotransfected with PPAR gamma and PPRE X 3-luciferase reporter construct. CLA effects on glucose tolerance and glucose homeostasis indicate that dietary CLA may prove to be an important therapy for the prevention and treatment of NIDDM.


Effect of conjugated linoleic acid on body composition in mice.

Park Y; Albright KJ; Liu W; Storkson JM; Cook ME; Pariza MW. Department of Food Microbiology and Toxicology, University of Wisconsin- Madison 53706, USA. Lipids 1997 Aug; 32(8): 853-8 PMID: 9270977 UI: 97417028

The effects of conjugated linoleic acid (CLA) on body composition were investigated. ICR mice were fed a control diet containing 5.5% corn oil or a CLA-supplemented diet (5.0% corn oil plus 0.5% CLA). Mice fed CLA- supplemented diet exhibited 57% and 60% lower body fat and 5% and 14% increased lean body mass relative to controls (P < 0.05). Total carnitine palmitoyltransferase activity was increased by dietary CLA supplementation in both fat pad and skeletal muscle; the differences were significant for fat pad of fed mice and skeletal muscle of fasted mice. In cultured 3T3-L1 adipocytes CLA treatment (1 x 10(-4)M) significantly reduced heparin-releasable lipoprotein lipase activity (- 66%) and the intracellular concentrations of triacylglyceride (-8%) and glycerol (- 15%), but significantly increased free glycerol in the culture medium (+22%) compared to control (P < 0.05). The effects of CLA on body composition appear to be due in part to reduced fat deposition and increased lipolysis in adipocytes, possibly coupled with enhanced fatty acid oxidation in both muscle cells and adipocytes.


Retention of conjugated linoleic acid in the mammary gland is associated with tumor inhibition during the post- initiation phase of carcinogenesis.

Ip C; Jiang C; Thompson HJ; Scimeca JA. Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA. Carcinogenesis 1997 Apr; 18(4): 755-9 PMID: 9111211 UI: 97265274

Conjugated linoleic acid (CLA) has been reported to have significant activity in inhibiting mammary carcinogenesis. A major objective of this study was to evaluate how changes in the concentration of CLA in mammary tissue as a function of CLA exposure/withdrawal were correlated with the rate of occurrence of mammary carcinomas. Rats treated with a single dose of dimethylbenz[a]anthracene (DMBA) at 50 days of age were given 1% CLA in the diet for either 4 weeks, 8 weeks or continuously following carcinogen administration. No cancer protection was evident in the 4 or 8 week-CLA treatment groups. Significant tumor inhibition was observed only in rats that were given CLA for the entire duration of the experiment (20 weeks). Analysis of CLA in the mammary gland showed that the incorporation of CLA was much higher in neutral lipids than in phospholipids. When CLA was removed from the diet, neutral lipid- and phospholipid-CLA returned to basal values in about 4 and 8 weeks, respectively. The rate of disappearance of neutral lipid-CLA (rather than phospholipid-CLA) subsequent to CLA withdrawal paralleled more closely the rate of occurrence of new tumors in the target tissue. It appears that neutral lipid-CLA may be a more sensitive marker of tumor protection than phospholipid-CLA. However, the physiological relevance of CLA accumulation in mammary lipids is unclear and remains to be determined. A secondary goal of this study was to investigate whether CLA might selectively inhibit clonal expansion of DMBA- initiated mammary epithelial cells with wild-type versus codon 61 mutated Ha-ras genes. Approximately 16% of carcinomas in the control group (without CLA) were found to express codon 61 ras mutation. Although continuous treatment with CLA reduced the total number of carcinomas by 70%, it did not alter the proportion of ras mutant versus wild-type carcinomas, suggesting that CLA inhibits mammary carcinogenesis irrespective of the presence or absence of the ras mutation.


Effects of dietary conjugated linoleic acid on lymphocyte function and growth of mammary tumors in mice.

Wong MW; Chew BP; Wong TS; Hosick HL; Boylston TD; Shultz TD. Department of Animal Sciences, Washington State University, Pullman 99164-6320, USA. Anticancer Res 1997 Mar-Apr; 17(2A): 987-93 PMID: 9137439 UI: 97283306

We studied the effects of conjugated linoleic acid (CLA) on lymphocyte function and growth of a transplantable murine mammary tumor. In experiment 1, eight-wk-old female Balb/c mice (n = 8/group) were fed 0.1%, 0.3% or 0.9% CLA for 3 or 6 wk. Lymphocyte proliferation, interleukin-2 production and lymphocyte cytotoxicity were assessed using splenic lymphocytes. Plasma CLA concentrations increased in a dose-dependent manner with CLA feeding. Lymphocyte proliferation in mice fed 0.3% and 0.9% CLA was enhanced in phytohemagglutinin-induced but not in concanavalin A- or lipopolysaccharide-stimulated cultures. Production of IL-2 also was stimulated by CLA. In contrast, CLA had no effect on lymphocyte cytotoxicity. In experiment 2, mice (n = 20/treatment) were fed the same diets for 2 wk before being infused with 1 x 10(6) WAZ-2T metastatic mammary tumor cells into the right inguinal mammary gland. Tumor volume and latency were recorded for 45 d. Dietary CLA did not affect mammary tumor growth. Tumor latency, tumor incidence and tumor lipid peroxidation activity also were unaffected by CLA. Body weight and feed intake were similar among treatments. Therefore, dietary CLA modulated certain aspects of the immune defense but had no obvious effect on the growth of an established, aggressive mammary tumor.


Conjugated linoleic acid suppresses the growth of human breast adenocarcinoma cells in SCID mice.


Visonneau S; Cesano A; Tepper SA; Scimeca JA; Santoli D; Kritchevsky D. Wistar Institute, Philadelphia, Pennsylvania 1910, USA. Anticancer Res 1997 Mar-Apr;17 (2A):969-73 PMID: 9137436 UI: 97283303

Conjugated linoleic acid (CLA), which is mainly derived from dairy products, has been shown both in vitro and in animal models to have strong anti-tumor activity. Particular effects were observed on the growth and metastatic spread of transplantable mammary tumors. In this study, we examined the effect of dietaryCLA on the growth of human breast adenocarcinoma cells in severe combined immunodeficient (SCID) mice. Mice were fed 1% CLA for two weeks prior to subcutaneous inoculation of 10(7) MDA-MB468 cells and throughout the study. Dietary CLA inhibited local tumor growth by 73% and 30% at 9 and 14 weeks post- inoculation, respectively. Moreover, CLA completely abrogated the spread of breast cancer cells to lungs, peripheral blood, and bone marrow. These results indicate the ability of dietary CLA to block both the local growth and systemic spread of human breast cancer via mechanisms independent of the host immune system.


Conjugated linoleic acid modulates hepatic lipid composition in mice.

Belury MA; Kempa-Steczko A. Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana 47907, USA. Lipids 1997 Feb; 32(2): 199-204 PMID: 9075211 UI: 97229214

Conjugated linoleic acid (CLA) is a chemoprotective fatty acid that inhibits mammary, colon, forestomach, and skin carcinogenesis in experimental animals. We hypothesize that the ubiquitous chemoprotective actions of dietary CLA in extrahepatic tissues are dependent upon its role in modulating fatty acid composition and metabolism in liver, the major organ for lipid metabolism. This study begins to evaluate the role of CLA in lipid metabolism by determining the modulation of fatty acid composition by CLA. Female SENCAR mice were fed semipurified diets containing 0.0% (Diet A), 0.5% (Diet B), 1.0% (Diet C), or 1.5% (Diet D) CLA (by weight) for six weeks. Mice fed Diets B, C, and D exhibited lower body weights and elevated amounts of extractable total lipid in livers compared with mice fed diets without CLA (Diet A). Analyses of the fatty acid composition of liver by gas chromatography revealed that dietary CLA was incorporated into neutral and phospholipids at the expense of linoleate in Diets B, C, and D; oleate increased and arachidonate decreased in neutral lipids of CLA diet groups. In addition, increasing dietary CLA was associated with reduced linoleate in hepatic phospholipids. In an in vitro assay, CLA was desaturated to an unidentified 18:3 product to a similar extent as linoleate conversion to gamma- linolenate (9.88, and 13.63%, respectively). These data suggest that CLA may affect metabolic interconversion of fatty acids in liver that may ultimately result in modified fatty acid composition and arachidonate-derived eicosanoid production in extrahepatic tissues. In addition to determining how dietary CLA modulates eicosanoid synthesis, further work is needed to identify enzymatic products that may result from desaturation of CLA.


Conjugated linoleic acid and linoleic acid are distinctive modulators of mammary carcinogenesis.

Ip C; Scimeca JA. Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA. Nutr Cancer 1997; 27(2): 131-5 PMID: 9121939 UI: 97191735

Previous work by Ip and co-workers showed that mammary cancer prevention by conjugated linoleic acid (CLA) is independent of the level of fat in the diet. Because CLA is an isomer of linoleic acid, there is the question regarding whether the effect of CLA is due to a displacement of linoleic acid in cells. To further evaluate whether there might be an interaction between linoleic acid and CLA, the present study was designed to examine the dose response to CLA (at 0.5%, 1%, 1.5%, and 2%) in rats fed a 2% or a 12% linoleate diet (both basal diets contained 20% total fat by weight). The end points of investigation included the bioassay of mammary tumorigenesis in the rat dimethylbenz[a]anthracene model as well as the incorporation of CLA, linoleic acid, and arachidonic acid in mammary glands. The mammary carcinogenesis results showed that the efficacy of tumor suppression by CLA was not affected by linoleate intake. With either linoleate diet, no further protection was evident with levels of CLA > 1%. Analysis of neutral lipids and phospholipids of the mammary tissue indicated that 1) the accumulation of CLA in mammary tissue was dose dependent from 0.5% to 2%, 2) CLA concentration was 10 times higher in neutral lipids than in phospholipids, 3) the incorporation of CLA in either fraction was not affected by the availability of linoleic acid, and 4) CLA did not appear to displace linoleic acid or arachidonic acid in the mammary tissue. The above findings suggest that there may be distinctive mechanisms in the modulation of tumor development by linoleic acid and CLA.


The efficacy of conjugated linoleic acid in mammary cancer prevention is independent of the level or type of fat in the diet.

Ip C; Briggs SP; Haegele AD; Thompson HJ; Storkson J; Scimeca JA. Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA. Carcinogenesis 1996 May; 17(5): 1045-50 PMID: 8640911 UI: 96217826

The objective of the present study was to investigate whether the anticarcinogenic activity of conjugated linoleic acid (CLA) is affected by the amount and composition of dietary fat consumed by the host. Because the anticancer agent of interest is a fatty acid, this approach may provide some insight into its mechanism of action, depending on the outcome of these fat feeding experiments. For the fat level experiment, a custom formulated fat blend was used that simulates the fatty acid composition of the US diet. This fat blend was present at 10, 13.3, 16.7 or 20% by weight in the diet. For the fat type experiment, a 20% (w/w) fat diet containing either corn oil (exclusively) or lard (predominantly) was used. Mammary cancer prevention by CLA was evaluated using the rat dimethylbenz[a]anthracene model. The results indicated that the magnitude of tumor inhibition by 1% CLA was not influenced by the level or type of fat in the diet. It should be noted that these fat diets varied markedly in their content of linoleate. Fatty acid analysis showed that CLA was incorporated predominantly in mammary tissue neutral lipids, while the increase in CLA in mammary tissue phospholipids was minimal. Furthermore, there was no evidence that CLA supplementation perturbed the distribution of linoleate or other fatty acids in the phospholipid fraction. Collectively these carcinogenesis and biochemical data suggest that the cancer preventive activity of CLA is unlikely to be mediated by interference with the metabolic cascade involved in converting linoleic acid to eicosanoids. The hypothesis that CLA might act as an antioxidant was also examined. Treatment with CLA resulted in lower levels of mammary tissue malondialdehyde (an end product of lipid peroxidation), but failed to change the levels of 8-hydroxydeoxyguanosine (a marker of oxidatively damaged DNA). Thus while CLA may have some antioxidant function in vivo in suppressing lipid peroxidation, its anticarcinogenic activity cannot be accounted for by protecting the target cell DNA against oxidative damage. The finding that the inhibitory effect of CLA maximized at 1% (regardless of the availability. of linoleate in the diet) could conceivably point to a limiting step in the capacity to metabolize CLA to some active product(s) which is essential for cancer prevention.


Dietary conjugated linoleic acid modulation of phorbol ester skin tumor promotion.

Belury MA; Nickel KP; Bird CE; Wu Y. Department of Foods and Nutrition, Purdue University, West Lafayette, IN 47907, USA. Nutr Cancer 1996; 26(2): 149-57 PMID: 8875552 UI: 97029542


The fatty acid derivative conjugated dienoic linoleate (CLA) has been shown to inhibit initiation and postinitiation stages of carcinogenesis in several experimental animal models. The goal of the present study was to determine the role of increasing levels of dietary CLA in mouse skin tumor promotion elicited by 12-O- tetradecanoylphorbol-13-acetate (TPA). Mice were fed control (no CLA) diet during initiation, then switched to diets containing 0.0%, 0.5%, 1.0%, or 1.5% (wt/wt) CLA during skin tumor promotion by TPA. Body weights of mice fed 0.5%, 1.0%, or 1.5% CLA were similar to each other but were significantly lower (p < 0.05) than weights of mice fed no CLA (0.0%) throughout promotion. A reduction in papilloma incidence was observed in mice fed 1.5% CLA from Weeks 8 to 24 compared with mice fed diets containing 0.0- 1.0% CLA (p < 0.05). Twenty-four weeks after tumor promotion was begun, diets containing 1.0% and 1.5% CLA inhibited tumor yield (4.94 and 4.35 tumors/mouse, respectively) compared with diets without CLA (0.0% CLA, 6.65 tumors/mouse, p < 0.05) or 0.5% CLA (5.92 tumors/mouse, p < 0.05). These data indicate that CLA inhibits tumor promotion in a manner that is independent of its anti-initiator activity. Further studies are warranted in identifying cellular mechanisms that are likely to be involved with the antipromoter effects of CLA.


Reinvestigation of the antioxidant properties of conjugated linoleic acid.

van den Berg JJ; Cook NE; Tribble DL Children's Hospital Oakland Research Institute, California 94609, USA. Lipids 1995 Jul; 30(7): 599-605 PMID: 7564914 UI: 96024076

Despite repeated suggestions that antioxidant activity of conjugated linoleic acid (CLA), a collective of conjugated dienoic isomers of linoleic acid, underlies its reported anticarcinogenic and antiatherosclerotic effects, the antioxidant properties of CLA remain ill-defined. Therefore, this study was undertaken to gain more insight into the mechanism of potential CLA antioxidant activity. It was tested whether CLA could protect membranes composed of 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC) from oxidative modification under conditions of metal ion- dependent or -independent oxidative stress. Progress of oxidation was determined by direct spectrophotometric measurement of conjugated diene formation and by gas chromatographic/mass spectrometric analysis of fatty acids. The oxidative susceptibility of CLA was higher than that of linoleic acid, and comparable to arachidonic acid. When oxidation of PLPC (1.0 mM) was initiated using the lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile) or the water- soluble 2,2'-azobis(2-amidinopropane) hydrochloride, the radical scavengers vitamin E and butylated hydroxytoluene (BHT) at 0.75 microM efficiently inhibited PLPC oxidation, as evident from a clear lag phase. In contrast, 0.75 microM CLA did not have any significant effect on PLPC oxidation. Inhibition of PLPC oxidation by higher concentrations of CLA appeared due to competition, not to an antioxidant effect. When oxidation of PLPC was initiated by hydrogen peroxide/Fe2+ (500 microM/0.05-20 microM), both vitamin E (1 microM) and ethylene glycol-bis(aminoethyl ether) tetraacetic acid (50 microM) efficiently inhibited PLPC oxidation. However, CLA (1-50 microM) did not show a clear protective effect under any of the conditions tested. We conclude that CLA, under these test conditions, does not act as an efficient radical scavenger in any way comparable to vitamin E or BHT. CLA also does not appear to be converted into a metal chelator under metal-ion dependent oxidative stress, as had previously been suggested. On the basis of our observations, a role for CLA as an antioxidant does not seem plausible.


Effect of timing and duration of dietary conjugated linoleic acid on mammary cancer prevention.

Ip C; Scimeca JA; Thompson H. Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA. Nutr Cancer 1995; 24(3): 241-7 PMID: 8610043 UI: 96193062

Conjugated linoleic acid (CLA) is a minor fatty acid found predominantly in the form of triglycerides in beef and dairy products. Previous work by Ip and co-workers showed that free fatty acid-CLA at < or = 1% in the diet is protective against mammary carcinogenesis in rats. The present study verified that the anticancer activities of free fatty acid-CLA and triglyceride-CLA are essentially identical. This is an important finding, because it rules out a nonspecific free fatty acid effect. In terms of practical implication, we can continue the in vivo research with the less-expensive free fatty acid-CLA without compromising the physiological relevance of the data. A primary objective of this report was to investigate how the timing and duration of CLA feeding might affect the development of mammary carcinogenesis in the methylnitrosourea (MNU) model. We found that exposure to 1% CLA during the early postweaning and pubertal period only (from 21 to 42 days of age) was sufficient to reduce subsequent tumorigenesis induced by a single dose of MNU given at 56 days of age. This period incidentally corresponds to a time of active morphological development of the mammary gland to the mature state. In contrast to the above observation, a continuous intake of CLA was required for maximal inhibition of tumorigenesis when CLA feeding was started after MNU administration, suggesting that some active metabolite(s) of CLA might be involved in suppressing the process of neoplastic promotion/progression.


Conjugated linoleic acid. A powerful anticarcinogen from animal fat sources.

Ip C; Scimeca JA; Thompson HJ. Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, New York 14263. Cancer 1994 Aug 1; 74(3 Suppl): 1050-4 PMID: 8039138 UI: 94313560

Conjugated linoleic acid (CLA) is a mixture of positional and geometric isomers of linoleic acid, which is found preferentially in dairy products and meat. Preliminary studies indicate that CLA is a powerful anticarcinogen in the rat mammary tumor model with an effective range of 0.1- 1% in the diet. This protective effect of CLA is noted even when exposure is limited to the time of weaning to carcinogen administration. The timing of this treatment corresponds to maturation of the mammary gland to the adult stage, suggesting that CLA may have a direct effect in reducing the cancer risk of the target organ. Of the vast number of naturally occurring substances that have been demonstrated to have anticarcinogenic activity in experimental models, all but a handful of them are of plant origin. Conjugated linoleic acid is unique because it is present in food from animal sources, and its anticancer efficacy is expressed at concentrations close to human consumption levels.


Conjugated linoleic acid and atherosclerosis in rabbits.

Lee KN; Kritchevsky D; Pariza MW. Department of Food Microbiology and Toxicology, University of Wisconsin- Madison 53706. Atherosclerosis 1994 Jul; 108(1): 19-25 PMID: 7980704 UI: 95071577

Conjugated linoleic acid (CLA) consists of a series of positional and geometric dienoic isomers of linoleic acid that occur naturally in foods. CLA exhibits antioxidant activity in vitro and in vivo. To assess the effect of CLA on atherosclerosis, 12 rabbits were fed a semi- synthetic diet containing 14% fat and 0.1% cholesterol for 22 weeks. For 6 of these rabbits, the diet was augmented with CLA (0.5 g CLA/rabbit per day). Blood samples were taken monthly for lipid analysis. By 12 weeks total and LDL cholesterol and triglycerides were markedly lower in the CLA-fed group. Interestingly, the LDL cholesterol to HDL cholesterol ratio and total cholesterol to HDL cholesterol ratio were significantly reduced in CLA-fed rabbits. Examination of the aortas of CLA-fed rabbits showed less atherosclerosis.


Potential of food modification in cancer prevention.

Ip C; Lisk DJ; Scimeca JA. Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, New York 14263. Cancer Res 1994 Apr 1; 54(7 Suppl): 1957s-1959s PMID: 8137319 UI: 94185053

This presentation focuses on research that could theoretically be applied to implement the strategy of general population chemoprevention. The concept is based on the premise of enhancing foods with known anticarcinogens through either agricultural methods or food- processing technologies. Two areas of our work are described: (a) garlic cultivated with selenium fertilization and (b) foods high in conjugated linoleic acid. Both selenium and conjugated linoleic acid are powerful chemopreventive agents in the animal tumor model. The rationale of delivering these two specific compounds through the food system will be developed. Preliminary studies will be summarized to show the feasibility of this approach in suppressing carcinogen-induced mammary cancer in rats. Finally, the advantages of using foods to provide anticarcinogens to the general population as part of a chemopreventive strategy will also be discussed.


Conjugated linoleic acid suppresses mammary carcinogenesis and proliferative activity of the mammary gland in the rat.

Ip C; Singh M; Thompson HJ; Scimeca JA. Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, New York 14263. Cancer Res 1994 Mar 1; 54(5): 1212-5 PMID: 8118808 UI: 94163610

Conjugated linoleic acid (CLA) is a collective term which refers to a mixture of positional and geometric isomers of linoleic acid. It is naturally occurring in meat and dairy products. We have previously reported (Ip, C., Chin, S. F., Scimeca, J. A., and Pariza, M. W. Cancer Res., 51: 6118-6124, 1991) that 1% CLA in the diet suppressed mammary carcinogenesis in rats given a high dose (10 mg) of 7,12- dimethylbenz(a)anthracene. In the present study, dietary CLA between 0.05 and 0.5% was found to produce a dose-dependent inhibition in mammary tumor yield when fed chronically to rats treated with a lower dose (5 mg) of 7,12-dimethylbenz(a)anthracene. Short-term CLA feeding for 5 weeks, from weaning to the time of carcinogen administration at 50 days of age, also offered significant protection against subsequent tumor occurrence. This period corresponds to maturation of the mammary gland to the adult stage in the rat. The inhibitory response to short- term CLA exposure was observed with the use of 2 different carcinogens: 7,12-dimethylbenz(a)anthracene and methylnitrosourea. The fact that CLA was protective in the methylnitrosourea model suggests that it may have a direct modulating effect on susceptibility of the target organ to neoplastic transformation. The proliferative activity of the mammary epithelium was assessed by the incorporation of bromodeoxyuridine. Immunohistochemical staining results showed that CLA reduced the labeling index of the lobuloalveolar compartment, but not that of the ductal compartment of the mammary tree. Since the lobuloalveolar structures are derived from the terminal end buds which are the sites of carcinogenic transformation, the above finding is consistent with the bioassay data of tumor inhibition. Thus, changes in gland development and morphogenesis may be a locus of action of CLA in modulating mammary carcinogenesis. CLA is a unique anticarcinogen because it is present in foods from animal sources. Furthermore, its efficacy in cancer protection is manifest at dietary concentrations which are close to the levels consumed by humans.


Feeding conjugated linoleic acid to animals partially overcomes catabolic responses due to endotoxin injection.

Miller CC; Park Y; Pariza MW; Cook ME. Poultry Science Dept., U.W. Madison 53706. Biochem Biophys Res Commun 1994 Feb 15; 198(3): 1107-12 PMID: 8117267 UI: 94161721

The ability of conjugated linoleic acid to prevent endotoxin-induced growth suppression was examined. Mice fed a basal diet or diet with 0.5% fish oil lost twice as much body weight after endotoxin injection than mice fed conjugated linoleic acid. By 72 hours post injection, mice fed conjugated linoleic acid had body weights similar to vehicle injected controls; however, body weights of basal and fish oil fed mice injected with endotoxin were reduced. Conjugated linoleic acid prevented anorexia from endotoxin injection. Splenocyte blastogenesis was increased by conjugated linoleic acid.


Differential stimulatory and inhibitory responses of human MCF-7 breast cancer cells to linoleic acid and conjugated linoleic acid in culture.

Shultz TD; Chew BP; Seaman WR. Department of Food Science and Human Nutrition, Washington State University, Pullman 99164-6376. Anticancer Res 1992 Nov-Dec; 12(6B): 2143-5 PMID: 1295460 UI: 93199244


Consumption of dietary fat has been linked to the high incidence of certain cancers. However, recent research has stimulated interest in conjugated linoleic acid (CLA), a newly recognized anticarcinogenic fatty acid. Human MCF-7 breast cancer cells were incubated for 12 d in culture medium supplemented with various concentrations (1.78-7.14 x 10(-5) M) of linoleic acid (LA) or CLA. Linoleic acid initially stimulated MCF-7 cell growth with an optimal effect at concentrations of 3.57-7.14 x 10(-5) M, but was inhibitory at similar concentrations after 8 and 12 d of incubation. In contrast, CLA was inhibitory to cancer cell growth at all concentrations and times tested. Cell growth inhibition by CLA was dose- and time-dependent. Growth retardation at the prescribed LA and CLA concentrations ranged, respectively, from 4 to 33% and 54 to 100% following 8 to 12 d of treatment. At similar LA and CLA concentrations, cytostatic and cytotoxic effects of CLA were more pronounced (8-81%) than LA. These in vitro results suggest that CLA is cytotoxic to MCF-7 cells.


Mammary cancer prevention by conjugated dienoic derivative of linoleic acid.

Ip C; Chin SF; Scimeca JA; Pariza MW. Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, New York 14263. Cancer Res 1991 Nov 15; 51(22): 6118-24 PMID: 1933874 UI: 92034729

Conjugated dienoic derivative of linoleic acid (CLA) is a collective term which refers to a mixture of positional and geometric isomers of linoleic acid. It is a naturally occurring substance in food and is present at higher concentrations in products from animal sources. The present study reports that synthetically prepared CLA is an effective agent in inhibiting the development of mammary tumors induced by dimethylbenz(a)anthracene. Rats were fed either the AIN-76A basal diet or the same diet supplemented with 0.5, 1, or 1.5% CLA by weight. These diets were started 2 weeks before carcinogen administration and continued until the end of the experiment. The total number of mammary adenocarcinomas in the 0.5, 1, and 1.5% CLA groups was reduced by 32, 56, and 60%, respectively. The final tumor incidence and cumulative tumor weight were similarly diminished in rats fed the CLA-containing diets. In general, there appeared to be a dose-dependent protection at levels of 1% CLA and below, but no further beneficial effect was evident at levels above 1%. Chronic feeding of up to 1.5% CLA produced no adverse consequences in the animals. Analysis of the phospholipid fraction from liver and mammary tumor extracts showed that only the c9,t11 isomer of CLA was incorporated and that the level of incorporation increased with dietary intake. An interesting property of CLA is its ability to suppress peroxide formation from unsaturated fatty acid in a test-tube model (Cancer Res., Ha et al. 50: 1097-1101, 1990). In view of this information, the amount of thiobarbituric acid- reactive substances (lipid peroxidation products) present endogenously in liver and mammary gland was quantitated. The feeding of CLA (for either 1 or 6 months) resulted in a decrease in the extent of lipid peroxidation in the mammary gland, but such a suppressive effect was not detected in the liver. It should be noted that maximal antioxidant activity was observed with only 0.25% CLA in the diet, whereas maximal tumor inhibition was achieved at about 1% CLA. Hence there is a discrepancy between the antioxidant efficacy of CLA and its anticarcinogenic potency, suggesting that some other mechanisms might be involved in cancer protection. Unlike the stimulatory effect of linoleic acid in carcinogenesis (Cancer Res., Ip et al., 45: 1997-2001, 1985), the reaction of CLA in cancer prevention is specific, and CLA is more powerful than any other fatty acid in modulating tumor development.


Inhibition of benzo(a)pyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid.

Ha YL; Storkson J; Pariza MW. Department of Food Microbiology and Toxicology, University of Wisconsin- Madison 53706. Cancer Res 1990 Feb 15; 50(4): 1097-101 PMID: 2297758 UI: 90124313

Grilled ground beef contains factors that inhibit the initiation of mouse epidermal carcinogenesis by 7,12- dimethylbenz(a)anthracene. Previously we isolated an active principal and characterized it as an isomeric mixture of conjugated dienoic derivatives of linoleic acid (CLA). We now show that synthetic CLA inhibits the initiation of mouse forestomach tumorigenesis by benzo(a) pyrene. Four and 2 days prior to p.o. treatment with benzo (a)pyrene, female ICR mice were given (a) CLA in olive oil, (b) linoleic acid in olive oil, or (c) olive oil alone or plus 0.85% saline (control groups). Three days later the cycle was repeated for a total of 4 times. At 30 wk of age, the mice were sacrificed. In three independent experiments, mice treated with CLA developed only about half as many neoplasms/animal as mice in the control groups (P less than 0.025); in two of the experiments tumor incidence was also reduced (P less than 0.05). There were no significant differences in food intake or body weight among the groups. High-performance liquid chromatography/gas chromatography analysis established that, following intubation, only the c-9, t-11 CLA isomer was incorporated into forestomach phospholipids. In studies aimed at elucidating the mechanism of action, we found that CLA is an effective antioxidant. Under the conditions of the test CLA was more potent than alpha- tocopherol and almost as effective as butylated hydroxytoluene. These observations indicate that CLA might serve as an in situ defense mechanism against membrane attack by free radicals and may, at least in part, explain the anticarcinogenic properties of CLA.


Conjugated dienoic derivatives of linoleic acid: a new class of anticarcinogens.

Pariza MW; Ha YL. Food Research Institute, University of Wisconsin, Madison 53706. Med Oncol Tumor Pharmacother 1990; 7(2-3): 169-71 PMID: 2232933 UI: 91041054

Evidence establishing the anticarcinogenic activity of the conjugated dienoic derivatives of linoleic acid (CLA) is reviewed. Our findings demonstrate that CLA is a potent antioxidant and that the c-9,t-11 CLA isomer is selectively incorporated into cellular phospholipid, which may at least in part explain the anticarcinogenic activity of CLA. Dietary sources of CLA include grilled beef, cheese, and related foods. Another source of CLA is its endogenous generation via the carbon centered free radical oxidation of linoleic acid. We propose that the formation and action of CLA represents a previously unrecognized in situ defense mechanism against membrane attack by oxygen radicals.