Final Report for LNC04-242
NE Iowa RC&D, Inc., Iowa State University, Coolee Region Organic Producers, and local producers joined to carry out a second project that will be a human feeding study using Conjugated Linoleic Acid (CLA) enriched food items verses non-CLA items. The purpose of this research will be to verify the health benefits of this acid in humans, to quantify how much CLA needs to be ingested to have an effect on health, as well as creating new practices for farmers to enhance the diets of their livestock to obtain an increased level of CLA in the final product.
Various isomers of conjugated octadecadienoic acid, also known as conjugated linoleic acid, have been cited as both improving health and inciting debilitating disorders. A wide variety of models have been used to determine the health implications of CLA consumption, including mice, rats, animal and human cell and tissue cultures, and humans. The methods of presenting CLA to the models have been just as varied. To date, only one study has investigated the effects of feeding dairy products high in CLA by dietary manipulation to humans (1). However, no studies have been conducted in which products naturally enriched with CLA have been fed to humans.
Feeding patterns and practices of ruminant livestock are known to alter the fatty acid composition of products derived from these animals. In particular, bovine animals consuming pasture during the rainy seasons tend to produce the highest natural concentrations of CLA (2).
One of many reported health effects of CLA is that of modulating lipoprotein profiles. Tricon et al have reported that in humans different isomers of CLA have opposing effects on circulating cholesterol concentrations: t10,c12 CLA improves lipoprotein profiles, whereas c9,t11 CLA deteriorates them (3). Other studies, however, have indicated that the c9,t11 isomer can decrease atherogenicity (4), and still others have shown no effect of CLA at all (1,5).
Another purported health benefits is hepatic health. In mouse and rat studies, CLA has been shown to decrease hepatic steatosis and alleviate the nonalcoholic fatty liver disease (6-8), though at least one study has concluded that CLA can be used to induce fatty liver (9). In humans, however, Iwata et al. showed little change in markers of liver function, with statistically significant changes being physiologically insignificant (10).
Other studies relate to improved and impaired insulin resistance and glucose intolerance (11,12), adiponectin modulation (11,13,14), and body composition (15,16). The benefits include decreasing the onset of diabetes symptoms and other markers of the metabolic syndrome, and the impairments have shown the exact opposite.
As briefly mentioned, different isomers may have different effects on health outcomes. Because no study has been done feeding the naturally occurring isomeric ratios in a natural food matrix such as that provided by pasture-fed cattle, the health effects are, for the most part, observations of synthetic, artificial, or manufactured products. With this in mind, the present study is designed to determine if human markers of health will be altered with the consumption of CLA in a natural food matrix.
This project will conduct an experiment that evaluates CLA-enriched dairy and beef foods from farmers on contract with CROPP. The farmers will use grazing systems to raise livestock and produce dairy products with high CLA content for the experiment. The milk and beef they produce will be processed and then prepared to create several different foods that will be incorporated into typical U.S. diets for a human feeding study. The study then will examine the health benefits of humans consuming CLA-enriched dairy and beef foods by evaluating the effect of feeding diets containing these CLA-enriched foods on concentration of cholesterol, individual lipoproteins, glucose, glucagon, and insulin in blood plasma, bone density, body weight and composition, and glucose tolerance. Results will be widely distributed through publication, field days and presentations. Several positive project outcomes are expected.
Short-term outcomes of the project will enhance the quality of life for participants and encourage sustainable agricultural systems.
Specific short-term outcomes include improved human health, quantified through the direct effects of CLA-enriched foods on measures of human health.
Because pasture feeding is the management system found to produce CLA-enriched foods, (outcome of our current SARE-funded project), intermediate outcomes of this research will improve the profitability for farmers that utilize sustainable management systems including greater economic viability for dairy and beef producers that use sustainable high CLA pasture systems. Once the benefit to humans is demonstrated, high CLA content meat and dairy products are expected to increase in market value. Therefore, producers using the sustainable, high CLA pasture system, will be able to obtain a premium price in new and emerging markets and increase the market share for high CLA content pasture-fed animals.
The long-term outcomes of this project will enhance the quality of life for farmers and ranchers, rural communities, and society as a whole by providing a better understanding of CLA as an indicator that links healthy lands and healthy animals to healthy humans.
Long-term benefits will include improved health of the land and of human communities. This will be accomplished through a demonstration of the economic, environmental, and health justifications of management systems that result in greater utilization of pasture-based feeding systems for production of animal-derived foods on marginal land.
Research was approved by the Iowa State University Institutional Review Board.
Women between the ages of 20 and 40, with a body mass index between 19 and 30, not pregnant or nursing, non-smoking, non- to moderate-drinkers, and who considered themselves in good health, were invited to be screened for participation. Screening included a fasting blood sample analyzed by Laboratory Corporation of America for serum triacylglycerols, total cholesterol, and HDL- cholesterol, determination of height, weight, and BMI, and a medical history questionnaire. Of the 56 women screened, 19 women fit the previously stated criteria and had serum cholesterol concentrations near or above the median for their age as determined by The Third National Health and Nutrition Examination Survey (NHANES III). One participant discontinued participation during the first weekend as a result of an injury unrelated to the study. The screened characteristics of the women who completed participation in the study are summarized in Table 1.
Diets were formulated by using Nutritionist Pro (version 2.3; First DataBank, Inc.; San Bruno, CA). Diets were formulated to average 2000 kcal/d with 35% of calories coming from lipid, 15% from protein, and 50% from carbohydrate, approximating the average values consumed by females age 20-40 y (17).
Beef with elevated concentrations of CLA was obtained by harvesting a pasture-fed beef steer after a period of high precipitation. The control ground beef was obtained from a feedlot-fed steer. Beef from both animals was ground into 85% lean ground beef.
CLA-enriched ice cream was obtained from Pastureland Creamery (Woodward, IA), CLA cheese from Farmers All Natural Creamery (Wellman, IA), and CLA-enriched butter was generously donated by PastureLand Cooperative (MN). Commercially available products were tested to find a control product similar to the treatment products in macronutrient composition. Haagen-Dazs ice cream, FastCo cheese, and Land-O-Lakes butter with typical CLA concentrations were chosen for the control products.
All meals were prepared in Iowa State University’s Human Metabolic Unit (HMU). Participants consumed 3 meals/d during weekdays and lunch on Sunday in the HMU. The other 5 weekly meals were prepared, packed out, and sent home with reheating instructions. Participants were asked to consume no other food or beverages than those provided except for water, tea, coffee, and no-calorie soft drinks sweetened with Splenda, for the duration of the 56-d intervention study.
Participants were asked to fast for at least 10 h prior to day 0. An intravenous catheter was inserted into the arm of each participant by registered nurses. Fasting serum samples were collected with serum separator tubes, and fasting plasma samples were collected in tubes treated with EDTA (Vacutainer, BD). An oral glucose tolerance test (OGTT) was conducted by administering a 75-g glucose drink (SUN-DEX, Fisher Scientific, No. TG30010P) that was to be consumed within five minutes. Blood again was drawn via IV catheter and placed into EDTA-treated tubes at 30, 60, 90, 120, 150, and 180 minutes after completion of the glucose drink.
Blood was allowed to coagulate at room temperature for 20-40 minutes for serum preparation. EDTA tubes for plasma preparation were kept on ice until centrifugation. Samples were centrifuged at 3000xg for 15 minutes at 4ºC. An aliquot of plasma was treated with aprotinin and stored in glass tubes for glucagon analysis. All other plasma and ther serum samples were aliquoted into plastic microfuge tubes and stored at -20ºC until analyzed.
Lipoproteins were isolated from 3 mL of “fasting” plasma sample by sequential flotation ultracentrifugation (18). Briefly, plasma was placed in a 4 mL polycarbonate tube and layered with a solution equal in density to the infranatant for a total volume of 3.9 mL. The sample was centrifuged at 109,000 xg for 20 hours at 18ºC in a Beckman Ti 50.4 rotor in a L8-M ultracentrifuge (Beckman, Palo Alto, CA); 1.8 mL of supernatant were transferred to a 2 mL Corning cryogenic vial. The infranatant then was adjusted to the next density, layered with the same density up to a volume of 3.9 mL, and the centrifugation procedure was repeated. The obtained fractions had densities of (g/mL): VLDL<1.006<IDL<1.019<LDL<1.063<HDL2<1.125<HDL3
Glucose was quantified colorimetrically in the OGTT samples (G7521Pointe Scientific). Insulin and glucagon were quantified in the OGTT samples by radioimmunoassay (HI-11K and GL-32K, respectively, Linco Research, St. Charles, MO), as were “fasting” concentrations of adiponectin (HADP-61HK, Linco Research).
Body composition was determined by using a Hologic Delphi W dual-energy x-ray absorptiometry (DXA) (Hologic Inc., Bedford, MA).
An extra day’s diets for each treatment were collected daily including all provided foods and liquids. Daily total intake was determined gravimetrically. Samples were stored at 4ºC until homogenized by using a Waring industrial blender with a cooling coil (Waring Commercial 4L Laboratory Blender, 4L 2610T coil, New Hartford, CT) set to 4ºC by a cryogenic cooler (Caron Model 2050, Marietta, OH). Homogenate was frozen at -20ºC until analyzed for composition.
Daily dry matter intake of each subject was determined by lyophilizing 5 replicates of 5-g aliquots of daily intake samples. Protein was approximated by using a micro-Kjeldahl procedure to determine nitrogen content. Briefly, 60±3 mg of the dry matter sample were added to Kjeldahl tubes in duplicate; a Kjeltab (Thompson and Capper Ltd, UK) and sulfuric acid were added to each tube. Samples were digested for 4 hours, and titrated to determine total nitrogen. Protein was approximated by using a factor of 6.25 times the nitrogen content.
Daily total lipid consumption was determined by a modified Folch procedure (19) with 2:1 chloroform:methanol (v:v). Lipids were extracted in triplicate, and lipid content was determined gravimetrically.
Fatty acid methyl esters (FAMEs) were prepared by using sodium methoxide as a methanolic base and quantified by a gas chromatograph. Initial column temperature was set to 70ºC and held for 2 min; increased to 165ºC at 19ºC/min and held for 20 min; increased to 192ºC at 0.6ºC/min and immediately increased to 230ºC at 50ºC/min and held for 15 min for a total run time of 88 min. Samples were analyzed on a Varian 3350 equipped with a Varian 8200CX autosampler and a Supelco 2560 fused silica capillary column (100 mx0.25 mmx0.2 μm film thickness). FAMEs were identified by retention times compared with purified lipid standards (NuChek, Elysian, MN).
Trimethylsilyl derivatives of sterols were prepared from the non-saponifiable fraction of one of the triplicates of the lipid extract. Briefly, 5α-cholestane was added as an internal standard and the lipid extract was saponified in 11% (w/v) potassium hydroxide in a 55% (v/v) ethanol/deionized water solution. The nonsaponifiable fraction was isolated and derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane. Sterol derivatives were analyzed on a 30 m column with an initial column temperature of 150ºC that was increased to 200ºC for 20 min followed by an increase to 250ºC for 30 minutes. Select sterol derivatives were identified by retention times compared with free sterols derivatized as described.
All statistics were analyzed by using the general linear model procedure of SAS (version 9,1,; SAS Institute Inc., Cary, NC). Values were compared as either the magnitude or percentage change from day 0 to day 56.
The composition of the diets was reasonably close to the target composition (Table 2). The lower macronutrient energy percentages of protein and lipid is likely an artifact of assuming that all dry matter was protein, lipid, or digestible carbohydrate, which results in an inflated carbohydrate approximation, overestimated total calories, and underestimated protein and lipid. However, for comparison purposes, the values obtained indicate a fairly similar dietary composition, with no differences in calculated intake of total energy, carbohydrate, and protein (all p>0.1), with the exception of a small (~2%), but statistically significant, difference in lipid percentage (p<0.05). Cholesterol intake was similar between the two diets.
Fatty acid profiles of the diets were similar in saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acids (PUFA) content (p>0.05), but with a significant difference in total CLA content and in specific isomers of CLA (p<0.0001) (Table 3). This resulted in an approximately 3.5-fold increase in CLA content in the CLA diet over the Control diet.
Changes in cholesterol concentrations in each lipoprotein fraction and fractions summed as the total cholesterol concentration recovered from the lipoprotein fractionation were analyzed (Figure 1A). All fractions were not different on day 0. Neither magnitude nor percentage changes in cholesterol concentrations from the beginning of the study to the end of the study between the two treatments were significantly different (p>0.05).
Triacylglycerol concentrations before and after the study were within normal ranges, with no significant differences at baseline. Changes from day 0 to day 56 were compared between the two diets (Figure 1B), with no significant differences (p>0.05) being observed.
Oral Glucose Tolerance
Plasma glucose, insulin, and glucagon concentrations were compared by using various measures of glucose intolerance including differences at each timepoint, as overall curves, as area-under-the-curve values (Figure 2A-C), and insulin resistance indices including homeostatic measurement of insulin resistance (data not shown). No markers of insulin resistance or glucose tolerance proved significant at p
Fasting adiponectin concentrations also were compared (Figure 2D). Fasting concentrations at day 0 were not significantly different. No significant differences in the percentage or magnitude change in adiponectin concentrations were observed between treatments.
The DXA scans of the left arm, right arm, trunk, left leg, right leg, and head were analyzed separately and as part of the subtotal (all parts except for the head) and total body compositions. No significant changes were noted in the separate body regions between the two treatments (data not shown). Of particular interest to health are subtotal and trunk compositions (Figure 3) to compare measures of whole body and abdominal, which also showed no significant differences between the two treatments (p>0.05).
Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured as markers of liver function (Figure 4). No differences were observed between the two treatments at baseline, and no differences were observed between the within treatment changes from day 0 to day 56.
(note: to view the tables from this report, contact the NCR-SARE office at 612-626-3113)
The focus of the present investigation was the comparison of the effects of naturally occurring CLA in beef and dairy products of commercial versus pasture-fed origin. However, there are a number of other dietary fatty acids with significant differences in concentration. In particular, the lipid percentages of identified odd-chain saturated and monounsaturated fatty acids were higher in the CLA diet than the Control diet. Little research has been done on these fatty acids with regard to human health. Furthermore, the ratio of identified n3:n6 fatty acids was significantly different analysis not shown, and particularly arachidonic acid (C20:4-n6), a precursor of inflammation-related prostaglandins, was lower in the CLA diet and α-linolenic acid (C18:3-n3), an essential fatty acid related to decreased cardio-vascular disease, was higher in the CLA diet. These confounding factors can never be removed from a study of this nature because of the intended design of using a complete diet with no dietary supplementation.
AST and ALT, when elevated in concentration, are indicative of hepatic dysfunction. A number of studies have shown that high consumption of CLA alter risk factors associated with liver dysfunction (6-9). However, the present study agrees with the results presented by Iwata et al. that no differences occur in human models(10), indicating that the concentrations of CLA consumed resulted in no physiologically significant changes in liver function.
No alteration of body composition was noted between the two treatments. Abdominal adiposity is generally more closely associated with the morbidity of the metabolic syndrome, and previous CLA studies have shown alterations in abdominal adiposity with CLA supplementation. Once again, our results indicate that no such effect can be seen between the diets administered.
Glucose intolerance and insulin resistance, two terms that are often interchanged, did not show any difference between the two treatments, whether the comparison used the entire timecourse or just the baseline homeostatic comparison.
Every measurement taken on the participants fed the CLA diet showed no significant changes when compared with the participants fed the Control diet. The inconsistency of these data with some of the literature can be explained in a number of ways. First, the daily intake of CLA was lower than what some researchers have shown necessary to effect a change. Second, if there is a dramatic isomeric effect, c9,t11 CLA may not be the active form of CLA, and the t10,c12 CLA was in very low abundance in the diets. Third, there were a number of differences in other fatty acids in the diet besides CLA; if CLA does have negative effects on health, as some studies have shown, perhaps the improvement of other fatty acids with purported health effects counterbalanced these changes. Finally, this study was meant to compare two realistic diets with 9 human subjects fed each diet. Other human studies have shown effects on free living humans substituting products at home or taking encapsulated products, which can result in a number of confounding factors such as compliance and unmeasured changes in food consumption desire and patterns. Furthermore, these methods are able to provide CLA intakes well above that found in a natural diet. Perhaps the inconsistency of our data with those studies is that the physiological concentration of CLA necessary to effect change cannot, as of yet, be achieved naturally, and the changes may not be seen when compared with an appropriate control.
The overall economic benefit of the project to the producer and the region remains positive with continued growth and interest from the public, consumers and producers. Because there are several opportunities for additional studies related to the benefits of human consumption of CLA through food products, this project has not inhibitated the positive impact of the overall study of CLA through Phase I and Phase II of the project.
Neither has it had a negative economic impact. Producers that adopted or expanded grass fed operations because of the over all project have continued to capture a portion of the beef and dairy market within the project area through direct sales and sales to restaurants and local food cooperatives. One of these restaurants has successfully integrated the grass-fed locally produced option for hamburgers on their menu at an additional charge, allowing them to pay more for the product to the producer and meet consumer demand for the product. Internet sales for these products from grass fed producers has also increased over the project period.
Several Northeast Iowa, Southeast Minnesota and Southwest Wisconsin beef and dairy producers with grass based systems had implemented or expanded their grass based systems after Phase I of the CLA project. They also had begun marketing their products as high CLA products to the public. In that marketing, they stressed the benefits of CLA as demonstrated in previous lab studies with non-human subjects to the consumers in their specific markets throughout the target area.
At the conclusion of this project, these producers felt that the Phase II study was relatively inconclusive. They noted the limited scope and duration of the study. They did not feel the results were significant enough for them to change the way they promote their grass based products. Their perception of the benefits of high CLA in beef and dairy products was not altered by the study. Since they have had a positive response to their systems and the marketing associated with it, they are not changing their early adoption of grass based systems and continue to promote the benefits of CLA based on early animal lab trials. Their markets continue to be strong.
During the period of time encompassed by this project - Phase II of the CLA project, grass system markets have been further strengthened by additional, outside factors and influences including a new push for locally grown products and an increasing desire from consumers to "know" their producers and the methods those producers use. The grass based systems previously adopted and increasingly being used throughout the project area provide an increasing number of multiple benefits and marketing opportunities.
Educational & Outreach Activities
Iowa State has committed to submitting this research to a nutrition-oriented journal (probably the American Journal of Clinical Nutrition) for publication. Other publications for this project include brochures and posters, which were utilized at field days, fairs and local foods conferences. Power point presentations were also utilized at workshops and conferences to mixed audiences of producers, consumers and policy makers.
1. Desroches, S., Chouinard, P. Y., Galibois, I., Corneau, L., Delisle, J., Lamarche, B., Couture, P. & Bergeron, N. (2005) Lack of effect of dietary conjugated linoleic acids naturally incorporated into butter on the lipid profile and body composition of overweight and obese men. Am J Clin Nutr 82: 309-319.
2. Noci, F., Monahan, F. J., French, P. & Moloney, A. P. (2005) The fatty acid composition of muscle fat and subcutaneous adipose tissue of pasture-fed beef heifers: influence of the duration of grazing. J Anim Sci 83: 1167-1178.
3. Tricon, S., Burdge, G. C., Kew, S., Banerjee, T., Russell, J. J., Jones, E. L., Grimble, R. F., Williams, C. M., Yaqoob, P. & Calder, P. C. (2004) Opposing effects of cis-9,trans-11 and trans-10,cis-12 conjugated linoleic acid on blood lipids in healthy humans. Am J Clin Nutr 80: 614-620.
4. Valeille, K., Ferezou, J., Amsler, G., Quignard-Boulange, A., Parquet, M., Gripois, D., Dorovska-Taran, V. & Martin, J. C. (2005) A cis-9,trans-11-conjugated linoleic acid-rich oil reduces the outcome of atherogenic process in hyperlipidemic hamster. Am J Physiol Heart Circ Physiol 289: H652-659.
5. Naumann, E., Carpentier, Y. A., Saebo, A., Lassel, T. S., Chardigny, J. M., Sebedio, J. L. & Mensink, R. P. (2006) Cis-9, trans- 11 and trans-10, cis-12 conjugated linoleic acid (CLA) do not affect the plasma lipoprotein profile in moderately overweight subjects with LDL phenotype B. Atherosclerosis 188: 167-174.
6. Noto, A., Zahradka, P., Yurkova, N., Xie, X., Nitschmann, E., Ogborn, M. & Taylor, C. G. (2006) Conjugated linoleic acid reduces hepatic steatosis, improves liver function, and favorably modifies lipid metabolism in obese insulin-resistant rats. Lipids 41: 179-188.
7. Zabala, A., Churruca, I., Macarulla, M. T., Rodriguez, V. M., Fernandez-Quintela, A., Martinez, J. A. & Portillo, M. P. (2004) The trans-10,cis-12 isomer of conjugated linoleic acid reduces hepatic triacylglycerol content without affecting lipogenic enzymes in hamsters. Br J Nutr 92: 383-389.
8. Nagao, K., Inoue, N., Wang, Y. M., Shirouchi, B. & Yanagita, T. (2005) Dietary conjugated linoleic acid alleviates nonalcoholic fatty liver disease in Zucker (fa/fa) rats. J Nutr 135: 9-13.
9. Yanagita, T., Wang, Y. M., Nagao, K., Ujino, Y. & Inoue, N. (2005) Conjugated linoleic acid-induced fatty liver can be attenuated by combination with docosahexaenoic acid in C57BL/6N mice. J Agric Food Chem 53: 9629-9633.
10. Iwata, T., Kamegai, T., Yamauchi-Sato, Y., Ogawa, A., Kasai, M., Aoyama, T. & Kondo, K. (2007) Safety of dietary conjugated linoleic acid (CLA) in a 12-weeks trial in healthy overweight Japanese male volunteers. J Oleo Sci 56: 517-525.
11. Purushotham, A., Wendel, A. A., Liu, L. F. & Belury, M. A. (2007) Maintenance of adiponectin attenuates insulin resistance induced by dietary conjugated linoleic acid in mice. J Lipid Res 48: 444-452.
12. Syvertsen, C., Halse, J., Hoivik, H. O., Gaullier, J. M., Nurminiemi, M., Kristiansen, K., Einerhand, A., O'Shea, M. & Gudmundsen, O. (2007) The effect of 6 months supplementation with conjugated linoleic acid on insulin resistance in overweight and obese. Int J Obes (Lond) 31: 1148-1154.
13. Zhou, X. R., Sun, C. H., Wang, H. Y., Jiang, L. Y. & Liu, R. (2005) [Effect of conjugated linoleic acid on gene expression of adiponectin of obese rat fed with high fat diet]. Zhonghua Yu Fang Yi Xue Za Zhi 39: 33-36.
14. Miller, J. R., Siripurkpong, P., Hawes, J., Majdalawieh, A., Ro, H. S. & McLeod, R. S. (2007) The trans-10, cis-12 Isomer of conjugated linoleic acid decreases adiponectin assembly by PPARgamma -dependent and PPARgamma -independent mechanisms. J Lipid Res.
15. Gaullier, J. M., Halse, J., Hoye, K., Kristiansen, K., Fagertun, H., Vik, H. & Gudmundsen, O. (2005) Supplementation with conjugated linoleic acid for 24 months is well tolerated by and reduces body fat mass in healthy, overweight humans. J Nutr 135: 778-784.
16. Gaullier, J. M., Halse, J., Hoye, K., Kristiansen, K., Fagertun, H., Vik, H. & Gudmundsen, O. (2004) Conjugated linoleic acid supplementation for 1 y reduces body fat mass in healthy overweight humans. Am J Clin Nutr 79: 1118-1125.
17. U.S. Department of Agriculture, A. R. S. (2007) Nutrient Intakes from Food: Mean Amounts and Percentages of Calories from Protein, Carbohydrate, Fat, and Alcohol, One Day, 2003-2004.
18. Havel, R. J., Eder, H. A. & Bragdon, J. H. (1955) The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 34: 1345-1353.
19. Folch, J., Lees, M. & Sloane Stanley, G. H. (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226: 497-509.
Areas needing additional study
At the onset of the project, the partners understood that because of funding and timeing limitations related to the grant, this project was a short term, limited study. The inconsistency of the results and study data with results from previous lab studies using CLA in a capsulated form rather than CLA within food products may be related to several factors including duration of the study, number of participants, or the ability of the participants to consume the needed levels of CLA from the prescribed diet. The levels of CLA within the food products could also be studied, be tested at even higher levels, or supplemented with additional CLA.
There were also some initial problems in obtaining the food products that were originally needed for the project. ISU used substitute food products from a fewer number of producers. Since it is currently unknown what the levels of CLA are in the beef and dairy products carried by various distributors and vendors, the control group consumed food products that may or may not have contained high CLA levels. NE Iowa RC&D feels that further evaluation is needed to consider all of these factors within each of these areas.