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Effects of conjugated linoleic acid on long term feeding in Fischer 344 rats
Food and Chemical Toxicology 43 (2005) 1273–1279 www.elsevier.com/locate/foodchemtox
Effects of conjugated linoleic acid on long term feeding in Fischer 344 rats Yeonhwa Park
a,*
, Karen J. Albright b, Michael W. Pariza
b
a
b
Department of Food Science, University of Massachusetts, 100 Holdsworth Way, Amherst, MA 01003, United States Food Research Institute, Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, WI 53706, United States Received 5 November 2004; accepted 16 February 2005
Abstract Weanling male Fischer 344 rats were fed either control or diet containing 1% CLA for 18 months. Weight gain and survival rate were not different between treatments, but CLA-fed animals ate slightly less food. CLA feeding did not significantly reduce body fat compared to that of control. Clinical chemistry and hematology analyses were performed on blood samples at week 69–72. CLA had no effects except on blood glucose, which was reduced in CLA-fed animals compared to control. All animals had chronic renal failure at the end of the study; however, CLA decreased the amount of protein in urine at week 70 of feeding. Necropsy and histopathology results indicated that there was no difference between treatment groups. Although this study used a limited number of animals and a single dose of CLA, our results suggest that long term CLA feeding did not cause any adverse effects in rats. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Conjugated linoleic acid; CLA; Rats; Glucose; Fischer; Proteinuria
1. Introduction Since the identification of conjugated linoleic acid (CLA) as an anticarcinogen from beef, it has been studied for a wide range of biological activity (Pariza et al., 2001). Among its activities are reducing development of atherosclerosis (Lee et al., 1994; Nicolosi et al., 1997), enhancing growth of animals (Chin et al., 1994), modulating immune responses (Miller et al., 1994; Cook et al., 1993; OÕShea et al., 2004), reducing type I hypersensitivity (Whigham et al., 2001, 2002), improving type II diabetes (Houseknecht et al., 1998; Belury et al., 2003), and reducing body fat while enhancing lean body mass (Park et al., 1997, 1999a; West et al., 1998, 2000; DeLany et al., 1999). It is believed that the multiple activities displayed by CLA may be the result of more than one iso-
*
Corresponding author. Tel.: +1 413 545 1018; fax: +1 413 545 1262. E-mail address: [email protected] (Y. Park).
0278-6915/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.02.015
mer present in the CLA preparation (Christie et al., 1997). Two major isomers, cis-9,trans-11 and trans10,cis-12 CLA, have been shown to be responsible for growth enhancement (Cook et al., 1999) and body fat reduction (Park et al., 1999b; de Deckere et al., 1999), respectively. Meanwhile, both of these isomers are reported to be effective in reducing carcinogenesis in the rat mammary model (Ip et al., 2000). With increased use of CLA as a dietary supplement, safety concerns over long-term use have to be addressed. Although most animal and human studies have not shown significant toxic effects by CLA, concerns of CLA use raised health related issues. With few exceptions most human clinical trials used durations less than 12 weeks. Recently Gaullier et al. (2004) reported on human trial in which no adverse effects were observed following one year CLA supplementation. Thus, the purpose of this study was to evaluate the effect of CLA in long term feeding (1.5 year or till death) in Fischer 344 rats.
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2. Materials and methods Weanling male Fischer 344 rats and semi-purified diet (TD94060, 99% basal mix) were purchased from Harlan Sprague-Dawley and Harlan Teklad, respectively, Madison, WI. CLA was obtained from Larodan Fine Chemicals AB (Malmo, Sweden) and its composition was 88.7% CLA (cis-9,trans-11, 41.9%; trans-10,cis-12, 43.5%; trans-9,trans-11/trans-10,trans-12, 1.5%; others, 1.8%) with the remainder as oleic acid, 5.6%; palmitic acid, 1.4%; linoleic acid, 0.5%; and other fatty acids, 3.8%. The diet was composed as follows (ingredient, g/kg): sucrose, 476; casein, vitamin-free test, 210; corn starch, 150; DL-methionine, 3; corn oil 60; cellulose, 50; mineral mix, AIN-76, 35; vitamin mix, AIN-76A, 10; calcium carbonate, 4; choline bitartrate, 2; and ethoxyquin, 0.1. Supplemental CLA (10 g/kg) was added to diets at the expense of corn oil. Diet was stored at 20 °C until use. Rats were housed individually in wire-bottomed cage in a windowless room with a 12-h light–dark cycle in strict accordance to guidelines established by the Research Animal Resources Center of University of Wisconsin-Madison. Diet and water, available ad libitum, were freshly provided twice a week. After a five-day adaptation period animals were randomly separated into groups. Ten rats were fed control diet and eleven were fed the diet supplemented with 1% CLA. In our previous work using mice we fed 0.5% CLA (Park et al., 1997, 1999a). Rats fed the same level did not respond greatly to CLA with regard to body fat reduction possibly due to the difference in the dose of CLA administered as a per weight basis (Park, 1996). 0.5% CLA in the diet would translate into approximately 0.85 g CLA/kg mice compared to 0.20–0.40 g CLA/kg rats (body weights of rats ranged from 200 to 400 g). Thus we chose 1% CLA for rat experiments in order to approximate the dose used in mice on a per weight basis. Body weight and food intake were measured weekly and twice a week, respectively. The number of animals to be used was determined for blood parameters and fat pad size, and thus is somewhat limited with regards to whole toxicological evaluation. Animals were observed closely and sacrificed if animals showed signs of discomfort, such as lethargy, anorexia, or loss of body weight (greater than 25% of their maximum weight). Four of control and three of CLA animals were died/sacrificed before the completion of the study (Fig. 1). At the end of the study, all animals were sacrificed with an overdose of sodium pentobarbital and examined for gross necropsy and histopathology by Dr. A.P. Gendron-Fitzpatrick (School of Veterinary Medicine, University of WisconsinMadison). After 12 weeks of feeding, three rats from each group were randomly selected to measure body fat. For body fat analyses, animals were sacrificed, gut contents were
Fig. 1. Survival curves. Male Fischer 344 rats were fed either control or 1% CLA containing diet for 81 weeks. Animals were observed closely during the feeding period and sacrificed if they showed signs of discomfort, such as lethargy, anorexia, or loss of body weight (greater than 25% of their maximum weight). Four control and three CLA animals were died/sacrificed out of seven for control and eight for CLA, respectively, before the completion of the study.
removed (to obtain empty carcass weight), and the carcasses (whole body) frozen at 20 °C. Frozen carcasses were chopped, and freeze dried to determine water content. Each dried carcass was ground to give a homogeneous sample. Carcass fat content was measured by
Table 1 Clinical chemistry and hematological analysesa Units
Blood urea nitrogen Creatinine Blood glucose Albumin AST/sGOT ALT Cholesterol Bilirubin Sodium Potassium White blood cell count Red blood cell count Hemoglobin Hematocrit Mean corpuscular volume Mean corpuscular Hb Mean corpuscular Hb conc. Red cell distribution width Platelet count Mean platelet volume
mg/dl mg/dl mg/dl g/dl IU/l IU/l mg/dl mg/dl mEq/l mEq/l 103/l 106/l g/dl % fl pg % (g/dl) % 103/l fl
Treatment Control
CLA
35.6 ± 5.5 0.70 ± 0.06 174.3 ± 1.9 2.9 ± 0.1 62.0 ± 6.0 46.5 ± 2.4 216.7 ± 16.3 0.33 ± 0.04 140.0 ± 0.9 3.92 ± 0.04 10.6 ± 1.4 7.58 ± 0.53 16.5 ± 0.8 41.6 ± 2.8 54.9 ± 0.4 21.8 ± 0.5 39.8 ± 1.0 20.1 ± 0.5 881 ± 28 6.3 ± 0.1
32.9 ± 5.7 0.66 ± 0.07 138.0b ± 4.7 3.2 ± 0.1 58.0 ± 4.1 45.6 ± 4.1 182.0 ± 8.8 0.40 ± 0.04 142.0 ± 1.0 4.30 ± 0.21 9.0 ± 1.3 7.44 ± 0.58 16.9 ± 0.8 42.2 ± 3.2 56.8b ± 0.4 21.5 ± 0.3 37.7 ± 0.5 18.6 ± 0.6 883 ± 69 6.6 ± 0.2
a Male Fischer 344 rats were fed either control or CLA containing diet (1%). Blood samples were taken from jugular vein at week 72 of feeding. Numbers are mean ± SE (n = 3–6 for control and n = 4–8 for CLA). b Indicates significantly different from control at P < 0.05 by Student t-test.
Y. Park et al. / Food and Chemical Toxicology 43 (2005) 1273–1279
extraction with diethyl ether overnight using a Soxhlet apparatus. For measuring fasting blood glucose, we made a slight incision lengthwise at the tip of the tail to get a blood sample after overnight fasting at three different days at week 69 and 70. Blood glucose was determined using a glucose analyzer (Sure Step, Model # L9116 GB 01114, Lifescan, Inc., Milpitas, CA). For glucose concentration of fed state, blood was drawn, between 9:30 and 11:00 am, from the jugular vein at week 72 and analyzed with Sigma Diagnostic Kit (Sigma Chemical Co., St Louis, MO). Plasma samples were also used for blood analysis (Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of Wisconsin-Madison). At week 70, water consumption was measured and urine samples were collected by placing animals in metabolic cages. The urine collected was used for urine protein analysis using Bio-Rad DC Protein assay kit.
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2.1. Statistics Data were analyzed with Student t-test for Tables 1 and 2 and Figs. 1–4 except fasted glucose in Fig. 4. Survival, necropsy, and histo-pathology data were analyzed with FisherÕs exact test using the Statistics Analysis System (SAS Users Guide: Statistics, SAS Institute Inc.,
Table 2 Tissue weights at necropsya Treatment (g)
Body weight Liver Lung Heart Kidney Adrenal gland Spleen Epididymal fat pad
Control
CLA
416.4 ± 17.3 13.3 ± 0.7 1.7 ± 0.2 1.23 ± 0.03 3.6 ± 0.1 0.091 ± 0.005 1.43 ± 0.10 13.3 ± 0.5
419.8 ± 6.9 13.9 ± 0.2 1.5 ± 0.1 1.30 ± 0.06 4.1 ± 0.2 0.101 ± 0.006 2.5 ± 0.67 14.4 ± 0.5
Fig. 3. Food intake. Male Fischer 344 rats were fed either control (open circles) or 1% CLA containing diet (filled circles). Numbers are mean ± SE (n = 10 for control and n = 11 for CLA up to week 12). Between week 13 and 69, n = 7 for control and n = 8 for CLA. From week 69, n = 3–7 for control and n = 5–8 for CLA. * indicates significantly different from control at that time point at P < 0.05 by Student t-test.
a Male Fischer 344 rats were fed either control or CLA containing diet (1%) for 81 weeks. Numbers are mean ± SE (n = 3 for control and n = 4 for CLA).
Fig. 2. Body weights. Male Fischer 344 rats were fed either control (open circles) or 1% CLA containing diet (filled circles). Numbers are mean ± SE (n = 10 for control and n = 11 for CLA up to week 12). Between week 13 and 69, n = 7 for control and n = 8 for CLA. From week 69, n = 3–7 for control and n = 5–8 for CLA. The only significant difference between control and CLA was observed at week 2 (p = 0.011).
Fig. 4. Concentration of blood glucose. Male Fischer 344 rats were fed either control (open bars) or 1% CLA containing diet (filled bars). Fasting blood glucose was measured at three different days at week 69 and 70. Glucose analyzer was used for a blood sample obtained by a slight incision lengthwise at the tip of the tail. For glucose concentration in the fed state, blood was drawn from the jugular vein at week 72 and plasma was used. Numbers are mean ± SE (n = 4–6 for control and n = 7–8 for CLA). * indicates significantly different from control at P < 0.05.
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Cary, NC). Since fasted plasma samples for glucose analysis were collected in three different days, data were analyzed with two-way ANOVA (treatments and collection days). Of major interest here are the comparisons among the treatments; these were computed using the Statistics Analysis System (SAS Users Guide: Statistics, SAS Institute Inc., Cary, NC) with the general linear mean procedure and least square means option. If the interaction between treatment and sample collection day was significant, this interaction was then used as the error term in the least square means analysis.
3. Results 3.1. Overall performance Animals were generally healthy. Fig. 1 shows the survival curves of these animals. The first sign of a health problem was observed as weight loss in one control animal at week 58, and it died at week 69. The cause of death was determined as pituitary tumor but it also had chronic renal disease. Based on our established symptoms of potential health problems (lethargy, anorexia and/or weight loss greater than 25% of animalÕs weight), three rats from each group were sacrificed before the completion of the study. All of these animals had severe chronic renal disease. Three animals of each group also had pituitary or testicular tumors. Necropsy details will be discussed later in this section. Although control animals tended to die earlier than CLA fed animals, this tendency was not statistically different. Further studies with a larger number of animals are needed to confirm what, if any, beneficial effects CLA supplementation has on survival. Figs. 2 and 3 show body weight and food intake, respectively. CLA-fed rats were slightly smaller than control but not significantly different. Food intake was lower in the CLA group compared to control. Increased food consumption for the last 15 weeks in control animals was due to wasting followed by anorexia and weight loss. This was also observed in other rats that were sacrificed before the completion of the study. 3.2. Body fat, urine analyses, clinical chemistry and hematology After 12 weeks of feeding, three rats of each group were sacrificed to measure body fat. Empty carcass weights and % water were not different between the groups (empty carcass weights were 303.5 g ± 2.6 for control and 302.6 g ± 4.6 for CLA, and water contents (%) were 58.3 ± 0.2 for control and 58.9 ± 0.4 for CLA). The percent body fat of CLA fed animals were less but not significantly different (14.1 ± 0.3 for control and 12.0 ± 0.9 for CLA).
Fig. 5. Protein in urine. Male Fischer 344 rats were fed either control or 1% CLA containing diet for 70 weeks. Numbers are mean ± SE (n = 5 for control and n = 8 for CLA). P = 0.06 by Student t-test.
Based on the observation that some animals tended to eat and drink more before weight loss, we speculated wasting syndrome such as diabetes or possibly kidney failure since the first animal that died had renal diseases. Thus we measured blood glucose, water consumption and protein from urine. CLA feeding significantly reduced blood glucose concentration in both fasted and fed animals (Fig. 4). Water consumption at week 70 was not different (23.7 ± 2.6 ml/23 h for control vs 20.8 ± 2.7 ml/23 h for CLA-fed rats). Protein was detected in the urine samples of both groups of animals, although CLA fed animals had less protein in urine (p = 0.06) compared to control (Fig. 5). Results of clinical chemistry and hematology showed all measurements were in the normal range except blood urea nitrogen (normal is 11–23 mg/dl) and cholesterol (normal is 44–138 mg/dl) (Table 1). Elevated levels of blood urea nitrogen in these animals (21–52 mg/dl) are indicative of renal function failure and elevated cholesterol level may be linked to age. There were no differences between the two treatment groups except blood glucose and mean corpuscular volume (MCV, Table 1). Since the difference of MCV between groups was significant but small, and since there was no difference in hematocrit between the two groups, the differences did not indicate any health concerns such as anemia. 3.3. Necropsy and histo-pathology Necropsy and histo-pathological examinations were performed on all animals. There were no differences in tissue weights between these two groups (Table 2). As mentioned earlier, all animals had chronic renal disease (such as chronic interstitial nephritis, nephrosis and/or glomerulosclerosis, Table 3). Pituitary tumors, testicular
Y. Park et al. / Food and Chemical Toxicology 43 (2005) 1273–1279 Table 3 Histopathologya Treatment
n Chronic renal diseasesb Prostatitis Testicular tumor or mass Pituitary tumor Lymphoma
Control
CLA
7 7 4 3 3 2
8 8 6 4 3 2
a Male Fischer 344 rats were fed either control or CLA containing diet (1%) for 81 weeks. Numbers indicate the number of animals with disease in each group. b Chronic interstitial nephritis, nephrosis and/or glomerulosclerosis.
tumors/masses, and/or prostatitis were observed in both treatment groups (Table 3). One of the CLA fed animals had an enlarged spleen, which was diagnosed as granular cell lymphoma. Also, two animals from each treatment had early stage granular cell lymphoma. Other disorders included gastritis, cardiomyopathy, focal hepatopathy, and interstitial pneumonitis, which were observed in one animal from each treatment group.
4. Discussion With recent interest in the use of CLA as a dietary supplement, its long term safety needs to be evaluated. A number of human trials have been reported so far with various results, from no effect to reduced body fat, fat free mass and/or weight reduction (Gaullier et al., 2004; Larsen et al., 2003; Jahreis et al., 2000; Riserus et al., 2003, 2004; Malpuech-Brugere et al., 2004; Kelley and Erickson, 2003; Terpstra, 2004). Most current CLA studies used relatively short term feeding except Gaullier et al. (2004), who reported a human trial with one year CLA supplementation. In this report, CLA decreased body fat mass without any adverse effects. Although it seems apparent that the effect of CLA on body fat reduction in humans is much less dramatic than in mice, evaluation of its safety is needed, especially for long-term use. This is the first report to evaluate long-term (1.5 year or till death) use of CLA in animals. Although this study used a limited number of animals and a single dose of CLA, our results suggest that CLA did not cause any adverse effects compared to control during this period. These results are consistent with Scimeca (1998), who reported that there was no toxicity related with feeding CLA (1.5% in the diet) to Fischer 344 rats for 36 weeks. Dietary CLA reduced body fat consistently in mice (Park et al., 1997, 1999a,b; West et al., 1998; DeLany et al., 1999) and pigs (Cook et al., 1998; Dugan et al., 2004), not only in growing animals but also in mature ones (Pariza et al., 2001). However, responses to CLA
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are not consistent in rats (Park, 1996) and humans (Larsen et al., 2003; Jahreis et al., 2000). Rats showed gender differences, with females responding better than males. Body fat of female SD rats was significantly reduced by CLA (23%) (Pariza et al., 1996), while male rats had very limited body fat reduction response to CLA (Park, 1996). We have performed five independent experiments with male rats (SD, Wistar Kyoto and Fischer; Park, 1996, this report and unpublished observation), and CLA slightly reduced body fat in all but one study (CLA reduced body fat 7% compared to control. Overall body fat (%) was 13.97 ± 0.48 for control and 13.04 ± 0.62 for CLA, p = 0.17, n = 29–30). Variable body fat response to CLA was also observed in human studies (Gaullier et al., 2004; Larsen et al., 2003; Jahreis et al., 2000; Malpuech-Brugere et al., 2004; Riserus et al., 2004; Eyjolfson et al., 2004). Different responses to CLA may be due to differences in metabolism. The fact that mice have a higher rate of fat turnover than other species may explain why CLA was most effective in mice. There has been concern over CLA use with regard to its effect of slight glucose tolerance in mice and humans (as observed by elevated blood glucose and insulin concentrations). This was correlated with the trans-10,cis-12 CLA isomer, but not with cis-9,trans-11 isomer or mixture of these two isomers (Riserus et al., 2001, 2002a,b; Tsuboyama-Kasaoka et al., 2000). We have previously suggested that CLA increased carnitine palmitoyltransferase activity in muscle (Park et al., 1997), which is the rate limiting enzyme for fatty acid b-oxidation. This implies that CLA can increase use of fatty acids as an energy source in muscle resulting in moderate glucose tolerance (Dumpke et al., 2000). However, in contrast to mice, CLA improved insulin sensitivity in Zucker diabetic fatty fa/fa rats (Houseknecht et al., 1998) as well as improved glucose concentration in Type 2 diabetes (Belury et al., 2003). OÕHagan and Menzel (2003) used 12% CLA and found transient increase of insulin in male rats. However, in the same study, with a lower dose, there was no difference in glucose or insulin levels. Our results indicate that 1% CLA supplementation may reduce glucose concentration. Even though the concentration of glucose in the current study is in the normal range for rats (80–300 mg/dl), the reduction of glucose observed in this study indicates CLA may help to reduce adult onset diabetes, often associated with aging. Liver, kidney, and adrenal gland hypertrophy associated with CLA were also reported by OÕHagan and Menzel (2003). This was only observed with 12% CLA feeding and was reversed by removal of CLA. Tsuboyama-Kasaoka et al. (2000) reported that CLA (1% of diet for six month) caused lipodystrophy in mice, which has the physiological symptoms of enlarged liver and small fat pad. The enlarged livers in CLA-fed mice were caused by the deposit of fat (Tsuboyama-Kasaoka et al.,
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2000; Belury and Kempa-Steczko, 1997). This has not observed in rats or pigs (Scimeca, 1998; Muller et al., 1999; Azain et al., 2000). We did not observe any differences in liver or kidney weights between the two groups in this study. Since mice, but not rats, responded well to fat reduction by CLA, it is possible that fat deposit in liver may be related with the reduction of fat in adipocytes. The chronic renal failure observed in all animals in our study may have been due to the high protein content of the diet compared to regular chow (14–16%). CLA feeding decreased the amount of protein in urine, which may indicate CLA decreased the severity of the renal failure. Similar findings have been reported (Yang et al., 2000), in which CLA feeding prolonged survival after development of proteinuria in NZB/W F1 mice. In summary, long term use of CLA, up to 18 months, did not cause any adverse effects in rats. CLA may reduce age-related diabetes as indicated by reduction of glucose concentration and may also improve survival after developing renal diseases. However, further toxicological evaluation for other species and a larger number of animals is required to ensure the safety and effectiveness of CLA, especially with regard to human consumption.
Acknowledgements We thank Ms. Jayne M. Storkson for assistance with manuscript preparation. This research was supported in part by gift funds administered through the University of Wisconsin-Madison Food Research Institute. Two of the authors (YP, MWP) are inventors of CLA use patents that are assigned to the Wisconsin Alumni Research Foundation.
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Y. Park et al. / Food and Chemical Toxicology 43 (2005) 1273–1279 OÕShea, M., Bassaganya-Riera, J., Mohede, I.C., 2004. Immunomodulatory properties of conjugated linoleic acid. Am. J. Clin. Nutr. 79, 1199S–1206S. Pariza, M., Park, Y., Cook, M., Albright, K., Liu, W., 1996. Conjugated linoleic acid (CLA) reduces body fat. FASEB J. 10, 3227. Pariza, M.W., Park, Y., Cook, M.E., 2001. The biologically active isomers of conjugated linoleic acid. Prog. Lipid Res. 40, 283–298. Park, Y., 1996. Regulation of energy metabolism and the catabolic effects of immune stimulation by conjugated linoleic acid. Ph.D. Thesis, University of Wisconsin-Madison. Park, Y., Albright, K.J., Liu, W., Storkson, J.M., Cook, M.E., Pariza, M.W., 1997. Effect of conjugated linoleic acid on body composition in mice. Lipids 32, 853–858. Park, Y., Albright, K.J., Storkson, J.M., Liu, W., Cook, M.E., Pariza, M.W., 1999a. Changes in body composition in mice during feeding and withdrawal of conjugated linoleic acid. Lipids 34, 243– 248. Park, Y., Storkson, J.M., Albright, K.J., Liu, W., Pariza, M.W., 1999b. Evidence that the trans-10,cis-12 isomer of conjugated linoleic acid induces body composition changes in mice. Lipids 34, 235–241. Riserus, U., Berglund, L., Vessby, B., 2001. Conjugated linoleic acid (CLA) reduced abdominal adipose tissue in obese middle-aged men with signs of the metabolic syndrome: a randomised controlled trial. Int. J. Obes. Relat. Metab. Disord. 25, 1129–1135. Riserus, U., Arner, P., Brismar, K., Vessby, B., 2002a. Treatment with dietary trans10cis12 conjugated linoleic acid causes isomer-specific insulin resistance in obese men with the metabolic syndrome. Diabetes Care 25, 1516–1521. Riserus, U., Basu, S., Jovinge, S., Fredrikson, G.N., Arnlov, J., Vessby, B., 2002b. Supplementation with conjugated linoleic acid causes isomer-dependent oxidative stress and elevated C-reactive protein: a potential link to fatty acid-induced insulin resistance. Circulation 106, 1925–1929. Riserus, U., Smedman, A., Basu, S., Vessby, B., 2003. CLA and body weight regulation in humans. Lipids 38, 133–137.
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Riserus, U., Smedman, A., Basu, S., Vessby, B., 2004. Metabolic effects of conjugated linoleic acid in humans: the Swedish experience. Am. J. Clin. Nutr. 79, 1146S–1148S. Riserus, U., Vessby, B., Arnlov, J., Basu, S., 2004. Effects of cis9,trans-11 conjugated linoleic acid supplementation on insulin sensitivity, lipid peroxidation, and proinflammatory markers in obese men. Am. J. Clin. Nutr. 80, 279–283. Scimeca, J.A., 1998. Toxicological evaluation of dietary conjugated linoleic acid in male Fischer 344 rats. Food Chem. Toxicol. 36, 391–395. Terpstra, A.H., 2004. Effect of conjugated linoleic acid on body composition and plasma lipids in humans: an overview of the literature. Am. J. Clin. Nutr. 79, 352–361. Tsuboyama-Kasaoka, N., Takahashi, M., Tanemura, K., Kim, H.J., Tange, T., Okuyama, H., Kasai, M., Ikemoto, S., Ezaki, O., 2000. Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice. Diabetes 49, 1534– 1542. West, D.B., Delany, J.P., Camet, P.M., Blohm, F., Truett, A.A., Scimeca, J., 1998. Effects of conjugated linoleic acid on body fat and energy metabolism in the mouse. Am. J. Physiol. 275, R667-72. West, D.B., Blohm, F.Y., Truett, A.A., DeLany, J.P., 2000. Conjugated linoleic acid persistently increases total energy expenditure in AKR/J mice without increasing uncoupling protein gene expression. J. Nutr. 130, 2471–2477. Whigham, L.D., Cook, E.B., Stahl, J.L., Saban, R., Bjorling, D.E., Pariza, M.W., Cook, M.E., 2001. CLA reduces antigen-induced histamine and PGE(2) release from sensitized guinea pig tracheae. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, R908-12. Whigham, L.D., Higbee, A., Bjorling, D.E., Park, Y., Pariza, M.W., Cook, M.E., 2002. Decreased antigen-induced eicosanoid release in conjugated linoleic acid-fed guinea pigs. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, R1104-12. Yang, M., Pariza, M.W., Cook, M.E., 2000. Dietary conjugated linoleic acid protects against end stage disease of systemic lupus erythematosus in the NZB/W F1 mouse. Immunopharmacol. Mmunotoxicol. 22, 433–449.
Effects of conjugated linoleic acid on long term feeding in Fischer 344 rats Yeonhwa Park
a,*
, Karen J. Albright b, Michael W. Pariza
b
a
b
Department of Food Science, University of Massachusetts, 100 Holdsworth Way, Amherst, MA 01003, United States Food Research Institute, Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, WI 53706, United States Received 5 November 2004; accepted 16 February 2005
Abstract Weanling male Fischer 344 rats were fed either control or diet containing 1% CLA for 18 months. Weight gain and survival rate were not different between treatments, but CLA-fed animals ate slightly less food. CLA feeding did not significantly reduce body fat compared to that of control. Clinical chemistry and hematology analyses were performed on blood samples at week 69–72. CLA had no effects except on blood glucose, which was reduced in CLA-fed animals compared to control. All animals had chronic renal failure at the end of the study; however, CLA decreased the amount of protein in urine at week 70 of feeding. Necropsy and histopathology results indicated that there was no difference between treatment groups. Although this study used a limited number of animals and a single dose of CLA, our results suggest that long term CLA feeding did not cause any adverse effects in rats. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Conjugated linoleic acid; CLA; Rats; Glucose; Fischer; Proteinuria
1. Introduction Since the identification of conjugated linoleic acid (CLA) as an anticarcinogen from beef, it has been studied for a wide range of biological activity (Pariza et al., 2001). Among its activities are reducing development of atherosclerosis (Lee et al., 1994; Nicolosi et al., 1997), enhancing growth of animals (Chin et al., 1994), modulating immune responses (Miller et al., 1994; Cook et al., 1993; OÕShea et al., 2004), reducing type I hypersensitivity (Whigham et al., 2001, 2002), improving type II diabetes (Houseknecht et al., 1998; Belury et al., 2003), and reducing body fat while enhancing lean body mass (Park et al., 1997, 1999a; West et al., 1998, 2000; DeLany et al., 1999). It is believed that the multiple activities displayed by CLA may be the result of more than one iso-
*
Corresponding author. Tel.: +1 413 545 1018; fax: +1 413 545 1262. E-mail address: [email protected] (Y. Park).
0278-6915/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.02.015
mer present in the CLA preparation (Christie et al., 1997). Two major isomers, cis-9,trans-11 and trans10,cis-12 CLA, have been shown to be responsible for growth enhancement (Cook et al., 1999) and body fat reduction (Park et al., 1999b; de Deckere et al., 1999), respectively. Meanwhile, both of these isomers are reported to be effective in reducing carcinogenesis in the rat mammary model (Ip et al., 2000). With increased use of CLA as a dietary supplement, safety concerns over long-term use have to be addressed. Although most animal and human studies have not shown significant toxic effects by CLA, concerns of CLA use raised health related issues. With few exceptions most human clinical trials used durations less than 12 weeks. Recently Gaullier et al. (2004) reported on human trial in which no adverse effects were observed following one year CLA supplementation. Thus, the purpose of this study was to evaluate the effect of CLA in long term feeding (1.5 year or till death) in Fischer 344 rats.
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2. Materials and methods Weanling male Fischer 344 rats and semi-purified diet (TD94060, 99% basal mix) were purchased from Harlan Sprague-Dawley and Harlan Teklad, respectively, Madison, WI. CLA was obtained from Larodan Fine Chemicals AB (Malmo, Sweden) and its composition was 88.7% CLA (cis-9,trans-11, 41.9%; trans-10,cis-12, 43.5%; trans-9,trans-11/trans-10,trans-12, 1.5%; others, 1.8%) with the remainder as oleic acid, 5.6%; palmitic acid, 1.4%; linoleic acid, 0.5%; and other fatty acids, 3.8%. The diet was composed as follows (ingredient, g/kg): sucrose, 476; casein, vitamin-free test, 210; corn starch, 150; DL-methionine, 3; corn oil 60; cellulose, 50; mineral mix, AIN-76, 35; vitamin mix, AIN-76A, 10; calcium carbonate, 4; choline bitartrate, 2; and ethoxyquin, 0.1. Supplemental CLA (10 g/kg) was added to diets at the expense of corn oil. Diet was stored at 20 °C until use. Rats were housed individually in wire-bottomed cage in a windowless room with a 12-h light–dark cycle in strict accordance to guidelines established by the Research Animal Resources Center of University of Wisconsin-Madison. Diet and water, available ad libitum, were freshly provided twice a week. After a five-day adaptation period animals were randomly separated into groups. Ten rats were fed control diet and eleven were fed the diet supplemented with 1% CLA. In our previous work using mice we fed 0.5% CLA (Park et al., 1997, 1999a). Rats fed the same level did not respond greatly to CLA with regard to body fat reduction possibly due to the difference in the dose of CLA administered as a per weight basis (Park, 1996). 0.5% CLA in the diet would translate into approximately 0.85 g CLA/kg mice compared to 0.20–0.40 g CLA/kg rats (body weights of rats ranged from 200 to 400 g). Thus we chose 1% CLA for rat experiments in order to approximate the dose used in mice on a per weight basis. Body weight and food intake were measured weekly and twice a week, respectively. The number of animals to be used was determined for blood parameters and fat pad size, and thus is somewhat limited with regards to whole toxicological evaluation. Animals were observed closely and sacrificed if animals showed signs of discomfort, such as lethargy, anorexia, or loss of body weight (greater than 25% of their maximum weight). Four of control and three of CLA animals were died/sacrificed before the completion of the study (Fig. 1). At the end of the study, all animals were sacrificed with an overdose of sodium pentobarbital and examined for gross necropsy and histopathology by Dr. A.P. Gendron-Fitzpatrick (School of Veterinary Medicine, University of WisconsinMadison). After 12 weeks of feeding, three rats from each group were randomly selected to measure body fat. For body fat analyses, animals were sacrificed, gut contents were
Fig. 1. Survival curves. Male Fischer 344 rats were fed either control or 1% CLA containing diet for 81 weeks. Animals were observed closely during the feeding period and sacrificed if they showed signs of discomfort, such as lethargy, anorexia, or loss of body weight (greater than 25% of their maximum weight). Four control and three CLA animals were died/sacrificed out of seven for control and eight for CLA, respectively, before the completion of the study.
removed (to obtain empty carcass weight), and the carcasses (whole body) frozen at 20 °C. Frozen carcasses were chopped, and freeze dried to determine water content. Each dried carcass was ground to give a homogeneous sample. Carcass fat content was measured by
Table 1 Clinical chemistry and hematological analysesa Units
Blood urea nitrogen Creatinine Blood glucose Albumin AST/sGOT ALT Cholesterol Bilirubin Sodium Potassium White blood cell count Red blood cell count Hemoglobin Hematocrit Mean corpuscular volume Mean corpuscular Hb Mean corpuscular Hb conc. Red cell distribution width Platelet count Mean platelet volume
mg/dl mg/dl mg/dl g/dl IU/l IU/l mg/dl mg/dl mEq/l mEq/l 103/l 106/l g/dl % fl pg % (g/dl) % 103/l fl
Treatment Control
CLA
35.6 ± 5.5 0.70 ± 0.06 174.3 ± 1.9 2.9 ± 0.1 62.0 ± 6.0 46.5 ± 2.4 216.7 ± 16.3 0.33 ± 0.04 140.0 ± 0.9 3.92 ± 0.04 10.6 ± 1.4 7.58 ± 0.53 16.5 ± 0.8 41.6 ± 2.8 54.9 ± 0.4 21.8 ± 0.5 39.8 ± 1.0 20.1 ± 0.5 881 ± 28 6.3 ± 0.1
32.9 ± 5.7 0.66 ± 0.07 138.0b ± 4.7 3.2 ± 0.1 58.0 ± 4.1 45.6 ± 4.1 182.0 ± 8.8 0.40 ± 0.04 142.0 ± 1.0 4.30 ± 0.21 9.0 ± 1.3 7.44 ± 0.58 16.9 ± 0.8 42.2 ± 3.2 56.8b ± 0.4 21.5 ± 0.3 37.7 ± 0.5 18.6 ± 0.6 883 ± 69 6.6 ± 0.2
a Male Fischer 344 rats were fed either control or CLA containing diet (1%). Blood samples were taken from jugular vein at week 72 of feeding. Numbers are mean ± SE (n = 3–6 for control and n = 4–8 for CLA). b Indicates significantly different from control at P < 0.05 by Student t-test.
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extraction with diethyl ether overnight using a Soxhlet apparatus. For measuring fasting blood glucose, we made a slight incision lengthwise at the tip of the tail to get a blood sample after overnight fasting at three different days at week 69 and 70. Blood glucose was determined using a glucose analyzer (Sure Step, Model # L9116 GB 01114, Lifescan, Inc., Milpitas, CA). For glucose concentration of fed state, blood was drawn, between 9:30 and 11:00 am, from the jugular vein at week 72 and analyzed with Sigma Diagnostic Kit (Sigma Chemical Co., St Louis, MO). Plasma samples were also used for blood analysis (Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of Wisconsin-Madison). At week 70, water consumption was measured and urine samples were collected by placing animals in metabolic cages. The urine collected was used for urine protein analysis using Bio-Rad DC Protein assay kit.
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2.1. Statistics Data were analyzed with Student t-test for Tables 1 and 2 and Figs. 1–4 except fasted glucose in Fig. 4. Survival, necropsy, and histo-pathology data were analyzed with FisherÕs exact test using the Statistics Analysis System (SAS Users Guide: Statistics, SAS Institute Inc.,
Table 2 Tissue weights at necropsya Treatment (g)
Body weight Liver Lung Heart Kidney Adrenal gland Spleen Epididymal fat pad
Control
CLA
416.4 ± 17.3 13.3 ± 0.7 1.7 ± 0.2 1.23 ± 0.03 3.6 ± 0.1 0.091 ± 0.005 1.43 ± 0.10 13.3 ± 0.5
419.8 ± 6.9 13.9 ± 0.2 1.5 ± 0.1 1.30 ± 0.06 4.1 ± 0.2 0.101 ± 0.006 2.5 ± 0.67 14.4 ± 0.5
Fig. 3. Food intake. Male Fischer 344 rats were fed either control (open circles) or 1% CLA containing diet (filled circles). Numbers are mean ± SE (n = 10 for control and n = 11 for CLA up to week 12). Between week 13 and 69, n = 7 for control and n = 8 for CLA. From week 69, n = 3–7 for control and n = 5–8 for CLA. * indicates significantly different from control at that time point at P < 0.05 by Student t-test.
a Male Fischer 344 rats were fed either control or CLA containing diet (1%) for 81 weeks. Numbers are mean ± SE (n = 3 for control and n = 4 for CLA).
Fig. 2. Body weights. Male Fischer 344 rats were fed either control (open circles) or 1% CLA containing diet (filled circles). Numbers are mean ± SE (n = 10 for control and n = 11 for CLA up to week 12). Between week 13 and 69, n = 7 for control and n = 8 for CLA. From week 69, n = 3–7 for control and n = 5–8 for CLA. The only significant difference between control and CLA was observed at week 2 (p = 0.011).
Fig. 4. Concentration of blood glucose. Male Fischer 344 rats were fed either control (open bars) or 1% CLA containing diet (filled bars). Fasting blood glucose was measured at three different days at week 69 and 70. Glucose analyzer was used for a blood sample obtained by a slight incision lengthwise at the tip of the tail. For glucose concentration in the fed state, blood was drawn from the jugular vein at week 72 and plasma was used. Numbers are mean ± SE (n = 4–6 for control and n = 7–8 for CLA). * indicates significantly different from control at P < 0.05.
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Cary, NC). Since fasted plasma samples for glucose analysis were collected in three different days, data were analyzed with two-way ANOVA (treatments and collection days). Of major interest here are the comparisons among the treatments; these were computed using the Statistics Analysis System (SAS Users Guide: Statistics, SAS Institute Inc., Cary, NC) with the general linear mean procedure and least square means option. If the interaction between treatment and sample collection day was significant, this interaction was then used as the error term in the least square means analysis.
3. Results 3.1. Overall performance Animals were generally healthy. Fig. 1 shows the survival curves of these animals. The first sign of a health problem was observed as weight loss in one control animal at week 58, and it died at week 69. The cause of death was determined as pituitary tumor but it also had chronic renal disease. Based on our established symptoms of potential health problems (lethargy, anorexia and/or weight loss greater than 25% of animalÕs weight), three rats from each group were sacrificed before the completion of the study. All of these animals had severe chronic renal disease. Three animals of each group also had pituitary or testicular tumors. Necropsy details will be discussed later in this section. Although control animals tended to die earlier than CLA fed animals, this tendency was not statistically different. Further studies with a larger number of animals are needed to confirm what, if any, beneficial effects CLA supplementation has on survival. Figs. 2 and 3 show body weight and food intake, respectively. CLA-fed rats were slightly smaller than control but not significantly different. Food intake was lower in the CLA group compared to control. Increased food consumption for the last 15 weeks in control animals was due to wasting followed by anorexia and weight loss. This was also observed in other rats that were sacrificed before the completion of the study. 3.2. Body fat, urine analyses, clinical chemistry and hematology After 12 weeks of feeding, three rats of each group were sacrificed to measure body fat. Empty carcass weights and % water were not different between the groups (empty carcass weights were 303.5 g ± 2.6 for control and 302.6 g ± 4.6 for CLA, and water contents (%) were 58.3 ± 0.2 for control and 58.9 ± 0.4 for CLA). The percent body fat of CLA fed animals were less but not significantly different (14.1 ± 0.3 for control and 12.0 ± 0.9 for CLA).
Fig. 5. Protein in urine. Male Fischer 344 rats were fed either control or 1% CLA containing diet for 70 weeks. Numbers are mean ± SE (n = 5 for control and n = 8 for CLA). P = 0.06 by Student t-test.
Based on the observation that some animals tended to eat and drink more before weight loss, we speculated wasting syndrome such as diabetes or possibly kidney failure since the first animal that died had renal diseases. Thus we measured blood glucose, water consumption and protein from urine. CLA feeding significantly reduced blood glucose concentration in both fasted and fed animals (Fig. 4). Water consumption at week 70 was not different (23.7 ± 2.6 ml/23 h for control vs 20.8 ± 2.7 ml/23 h for CLA-fed rats). Protein was detected in the urine samples of both groups of animals, although CLA fed animals had less protein in urine (p = 0.06) compared to control (Fig. 5). Results of clinical chemistry and hematology showed all measurements were in the normal range except blood urea nitrogen (normal is 11–23 mg/dl) and cholesterol (normal is 44–138 mg/dl) (Table 1). Elevated levels of blood urea nitrogen in these animals (21–52 mg/dl) are indicative of renal function failure and elevated cholesterol level may be linked to age. There were no differences between the two treatment groups except blood glucose and mean corpuscular volume (MCV, Table 1). Since the difference of MCV between groups was significant but small, and since there was no difference in hematocrit between the two groups, the differences did not indicate any health concerns such as anemia. 3.3. Necropsy and histo-pathology Necropsy and histo-pathological examinations were performed on all animals. There were no differences in tissue weights between these two groups (Table 2). As mentioned earlier, all animals had chronic renal disease (such as chronic interstitial nephritis, nephrosis and/or glomerulosclerosis, Table 3). Pituitary tumors, testicular
Y. Park et al. / Food and Chemical Toxicology 43 (2005) 1273–1279 Table 3 Histopathologya Treatment
n Chronic renal diseasesb Prostatitis Testicular tumor or mass Pituitary tumor Lymphoma
Control
CLA
7 7 4 3 3 2
8 8 6 4 3 2
a Male Fischer 344 rats were fed either control or CLA containing diet (1%) for 81 weeks. Numbers indicate the number of animals with disease in each group. b Chronic interstitial nephritis, nephrosis and/or glomerulosclerosis.
tumors/masses, and/or prostatitis were observed in both treatment groups (Table 3). One of the CLA fed animals had an enlarged spleen, which was diagnosed as granular cell lymphoma. Also, two animals from each treatment had early stage granular cell lymphoma. Other disorders included gastritis, cardiomyopathy, focal hepatopathy, and interstitial pneumonitis, which were observed in one animal from each treatment group.
4. Discussion With recent interest in the use of CLA as a dietary supplement, its long term safety needs to be evaluated. A number of human trials have been reported so far with various results, from no effect to reduced body fat, fat free mass and/or weight reduction (Gaullier et al., 2004; Larsen et al., 2003; Jahreis et al., 2000; Riserus et al., 2003, 2004; Malpuech-Brugere et al., 2004; Kelley and Erickson, 2003; Terpstra, 2004). Most current CLA studies used relatively short term feeding except Gaullier et al. (2004), who reported a human trial with one year CLA supplementation. In this report, CLA decreased body fat mass without any adverse effects. Although it seems apparent that the effect of CLA on body fat reduction in humans is much less dramatic than in mice, evaluation of its safety is needed, especially for long-term use. This is the first report to evaluate long-term (1.5 year or till death) use of CLA in animals. Although this study used a limited number of animals and a single dose of CLA, our results suggest that CLA did not cause any adverse effects compared to control during this period. These results are consistent with Scimeca (1998), who reported that there was no toxicity related with feeding CLA (1.5% in the diet) to Fischer 344 rats for 36 weeks. Dietary CLA reduced body fat consistently in mice (Park et al., 1997, 1999a,b; West et al., 1998; DeLany et al., 1999) and pigs (Cook et al., 1998; Dugan et al., 2004), not only in growing animals but also in mature ones (Pariza et al., 2001). However, responses to CLA
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are not consistent in rats (Park, 1996) and humans (Larsen et al., 2003; Jahreis et al., 2000). Rats showed gender differences, with females responding better than males. Body fat of female SD rats was significantly reduced by CLA (23%) (Pariza et al., 1996), while male rats had very limited body fat reduction response to CLA (Park, 1996). We have performed five independent experiments with male rats (SD, Wistar Kyoto and Fischer; Park, 1996, this report and unpublished observation), and CLA slightly reduced body fat in all but one study (CLA reduced body fat 7% compared to control. Overall body fat (%) was 13.97 ± 0.48 for control and 13.04 ± 0.62 for CLA, p = 0.17, n = 29–30). Variable body fat response to CLA was also observed in human studies (Gaullier et al., 2004; Larsen et al., 2003; Jahreis et al., 2000; Malpuech-Brugere et al., 2004; Riserus et al., 2004; Eyjolfson et al., 2004). Different responses to CLA may be due to differences in metabolism. The fact that mice have a higher rate of fat turnover than other species may explain why CLA was most effective in mice. There has been concern over CLA use with regard to its effect of slight glucose tolerance in mice and humans (as observed by elevated blood glucose and insulin concentrations). This was correlated with the trans-10,cis-12 CLA isomer, but not with cis-9,trans-11 isomer or mixture of these two isomers (Riserus et al., 2001, 2002a,b; Tsuboyama-Kasaoka et al., 2000). We have previously suggested that CLA increased carnitine palmitoyltransferase activity in muscle (Park et al., 1997), which is the rate limiting enzyme for fatty acid b-oxidation. This implies that CLA can increase use of fatty acids as an energy source in muscle resulting in moderate glucose tolerance (Dumpke et al., 2000). However, in contrast to mice, CLA improved insulin sensitivity in Zucker diabetic fatty fa/fa rats (Houseknecht et al., 1998) as well as improved glucose concentration in Type 2 diabetes (Belury et al., 2003). OÕHagan and Menzel (2003) used 12% CLA and found transient increase of insulin in male rats. However, in the same study, with a lower dose, there was no difference in glucose or insulin levels. Our results indicate that 1% CLA supplementation may reduce glucose concentration. Even though the concentration of glucose in the current study is in the normal range for rats (80–300 mg/dl), the reduction of glucose observed in this study indicates CLA may help to reduce adult onset diabetes, often associated with aging. Liver, kidney, and adrenal gland hypertrophy associated with CLA were also reported by OÕHagan and Menzel (2003). This was only observed with 12% CLA feeding and was reversed by removal of CLA. Tsuboyama-Kasaoka et al. (2000) reported that CLA (1% of diet for six month) caused lipodystrophy in mice, which has the physiological symptoms of enlarged liver and small fat pad. The enlarged livers in CLA-fed mice were caused by the deposit of fat (Tsuboyama-Kasaoka et al.,
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2000; Belury and Kempa-Steczko, 1997). This has not observed in rats or pigs (Scimeca, 1998; Muller et al., 1999; Azain et al., 2000). We did not observe any differences in liver or kidney weights between the two groups in this study. Since mice, but not rats, responded well to fat reduction by CLA, it is possible that fat deposit in liver may be related with the reduction of fat in adipocytes. The chronic renal failure observed in all animals in our study may have been due to the high protein content of the diet compared to regular chow (14–16%). CLA feeding decreased the amount of protein in urine, which may indicate CLA decreased the severity of the renal failure. Similar findings have been reported (Yang et al., 2000), in which CLA feeding prolonged survival after development of proteinuria in NZB/W F1 mice. In summary, long term use of CLA, up to 18 months, did not cause any adverse effects in rats. CLA may reduce age-related diabetes as indicated by reduction of glucose concentration and may also improve survival after developing renal diseases. However, further toxicological evaluation for other species and a larger number of animals is required to ensure the safety and effectiveness of CLA, especially with regard to human consumption.
Acknowledgements We thank Ms. Jayne M. Storkson for assistance with manuscript preparation. This research was supported in part by gift funds administered through the University of Wisconsin-Madison Food Research Institute. Two of the authors (YP, MWP) are inventors of CLA use patents that are assigned to the Wisconsin Alumni Research Foundation.
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