Evaluation of Health Benefits of Conjugated Linoleic Acid (CLA)-Enriched Dairy and Beef Foods

Evaluation of Health Benefits of Conjugated Linoleic Acid (CLA)-Enriched Dairy and Beef Foods

Summary

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 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.

Objectives/Performance Targets

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.

Accomplishments/Milestones

General Introduction

The researchers at Iowa State University contracted to perform the research study entitled “Evaluation of health benefits of conjugated linoleic acid (CLA) — enriched dairy and beef foods” have worked arduously at generating the most comprehensive and accurate representation of the results CLA can have on human health. In the following update, the block from the fall of 2005 is reviewed, the attempted block in the summer of 2006 is addressed, and the current activities and plans for the spring 2006 block are outlined. Also, the efforts of the researchers at optimizing the recruitment and retention of subjects is reflected through a short study on sucralose, and attention has been given to delivering the greatest amount of CLA to the subjects as possible through beef and dairy products. In all, the efforts thus far guarantee a successful and accurate study in the spring of 2007.

Fall 2005 – Human Subjects Approval
The study was approved by the Iowa State University Institutional Review Board (IRB), the committee in charge of approving human subjects research, on April 19th 2005, at which point recruitment and study preparation could begin.

Recruitment:
Over 10,000 women were contacted for participation through email, fliers, and handouts. Of the women contacted, approximately 80 women agreed to be screened for participation, though only about 40 women attended the screening day. Screening for participation included signing an informed consent document (Appendix A), a brief medical history questionnaire (Appendix B), height and weight measurements for BMI calculation, and a blood sample. The blood samples were sent to LabCorp, a clinical laboratory, for analysis. An example of the results obtained from LabCorp is shown in Appendix C. After review of the potential participants, only 13 were found to have cholesterol in a range that could be considered elevated. These women were invited to participate in the first block of a two block study. The screening parameters from the women that completed the study are shown in Table 1.
(table 1 on hard copy only)

Treatment foods:
Two classes of experimental foods were obtained: control, which is comprised of feedlot beef and dairy products, and treatment, which is comprised of pasture or grassfed beef and dairy products.
The feedlot beef was obtained from the Iowa State University Meat Labs. The meat was ground into 85% lean ground beef. The dairy products are obtained from commercial sources known to use only feedlot cattle.

The treatment foods were obtained from various sources. Originally, the milk for the dairy products, including ice cream, cheese and butter, was to be obtained from pasture and grassfed producers in north east Iowa. Milk was tested from these producers, and one, Don Baker, was selected from which to purchase the milk. A representative from Organic Valley in LaFarge, WI, was to coordinate the collection of the milk and its subsequent processing into the final dairy products mentioned previously. The milk was transported to a dairy in Wellman, IA, that was not outfitted to make the small batches requested by the representative from Organic Valley. The milk that was transported was subsequently processed into over 200 lbs of soft cheddar cheese, leaving the ice cream and the butter to be obtained elsewhere. The time at which these problems occurred was such that another collection from Mr. Baker, as well as locating another processing plant, was not feasible if the study was to start on time. Therefore, existing pasture and grass fed operations were sought after for the ice cream and the butter. The ice cream was purchased from Picket Fence Creamery, a one family pasture fed operation out of Woodward, IA. Butter was purchased from Pastureland in Minnesota.

All foods were stored at 20˚C in a controlled walk-in freezer, which allows properly packaged food to stay fresh and appropriate for human consumption for at least two years.

Feeding Study:
Twelve subjects were evenly divided between two groups, six in the control group and six in the treatment group. On the first day, one subject dropped out for personal reasons unrelated to the study. Her alternate was contacted, and was also unable to participate for personal reasons unrelated to the study. On the third day, a subject dropped out because she found the protocol too strenuous for her lifestyle. This left four participants in the treatment group and six in the control group. All of these participants completed the study successfully.

Blood was taken every two weeks, with an oral glucose tolerance test (OGTT) being administered on weeks 0 and 8. Each time point included one blood collection tube which was sent to LabCorp, a clinical laboratory, and another was retained for in lab analysis.

On weeks 0 and 8, participants were analyzed with dual-energy x-ray absorptiometry (DXA) for bone density and body composition, an example of which is shown in Appendix D.

Results:
The results from LabCorp were analyzed for differences between weeks 0 and 8 for total cholesterol (TC), triacylglycerols (TAG), high-density lipoprotein cholesterol (HDL-C), a very-low-density lipoprotein calculation (VLDL) and a low-density lipoprotein cholesterol calculation (LDL-C) (Table 2). VLDL is directly calculated from TAG by dividing the concentration of TAG by five. LDL-C is calculated by subtracting the concentration of HDL-C and the calculated concentration of VLDL from the concentration of TC. There appeared to be a trend toward the treatment group having decreased serum TC (p=0.1396). However, this trend appeared to be primarily from the significant decrease in serum HDL-C in the treatment group (p=0.0350). VLDL and TAG did not change significantly between the two groups (p=0.2896 for both because VLDL is calculated from TAG), nor did the treatment cause a change in LDL-C concentrations (p=0.6676).

Blood glucose and insulin concentrations are currently being measured for the OGTT. Calculations will be done regarding these concentrations to determine insulin resistance and insulin sensitivity.

Discussion:
In-depth analysis was not conducted on the samples from this trial because of differences in macronutrient compositions in the foods. According to our laboratory analyses, the experimental foods were comprised of macronutrient compositions that differed from what the producers had indicated. For instance, one form of ground beef contained approximately 15% lipid, while the other contained only 6%.

Furthermore, the technique used to determine the lipid profiles of the products was not optimized for the detection of conjugated linoleic acid. The macronutrient compositions of the foods are being reanalyzed to determine if the disparities were the result of the analysis or the result of the products themselves.

(table 2 on hard copy only)
The glucose tolerance test is being analyzed to make use of reagents presently in the lab before they expire, meaning that the tests are being conducted with little or no cost to the project.
Furthermore, the results from LabCorp were analyzed statistically, disregarding any differences in the results that may have been the product of the differences in diet composition.

At this point, we can conclude that the treatment diet tended to cause a decrease in total cholesterol primarily as the product of a decrease in the HDL cholesterol as compared to the control group.

Summer 2006:
A second block of the study was planned for the summer months of 2006. An updated IRB form was submitted in March 2006 for the annual review. The updated informed consent document and medical history questionnaire are attached as Appendices E and F, respectively. The updated IRB form contained needed changes that became apparent during the first block. The IRB committee, however, did not approve the study until April 19th, 2006, though the announcement of approval was withheld until the IRB committee confirmed approval of the use of the radiation from DXA that was required by the state. Announcement of approval came in May. Without IRB approval, potential subjects were unable to sign informed consent documents, and therefore recruitment was stunted. Recruitment efforts continued after the approval, but the time frame was such that the recruitment campaign only succeeded in recruiting a handful of participants, which did not warrant proceeding with the study.

Current Activity and Plans for Spring 2007

Recruitment:
The IRB approval for the attempted summer block is still approved through April 2007. Recruitment was started for spring 2007 when students returned for classes in September 2006. Over 7,000 women between the ages of 20 and 40 were contacted via email, fliers, word of mouth, presentation, and posters. Several hundred women responded, indicating an interest in the study. Of these, 60 women were interested in coming in to get screened, though only 41 came in to be screened. This screening group included women that were screened in the summer block who were found to have cholesterol in a range that qualified them for the study. 22 of the 41 women screened have cholesterol of 160 mg/dL or above, which is our present qualifying range. We have several more women scheduled to be screened, from which we hope to find enough women to have over 24 qualifying participants, so as to also have alternates.

Experimental Foods:
The beef from the previous study was sealed and stored in a manner that will make it able to be used in the upcoming block, without concerns of flavor or food integrity. The treatment butter and cheese will also be able to be used. New ice cream was purchased in late summer and is stored at -20˚C in the same freezer as the beef, butter, and cheese. Control butter, cheese, and ice cream are to be purchased prior to the start of the feeding study.

The foods are currently being reanalyzed to confirm their macronutrient compositions, as well as their lipid profiles and cholesterol concentrations. Previous analyses resulted in significant differences between the two treatment food groups in macronutrient and cholesterol concentrations, and the lipid analysis was not optimized for detection and quantification of CLA. These composition determinations are not only important in analyzing the first block, but also in guaranteeing the success of the block in the spring of 2007. The foods need to be as similar as possible except for the lipid profiles to create a properly controlled experiment.

Feeding Study:
The only fundamental change from the first block to the second block in regards to the human subject portion of the research is a more comprehensive OGTT. This test will require participants to have an intravenous catheter inserted, rather than multiple needle sticks, so as to collect a blood sample before consumption of the glucose drink and 30, 60, 90, 120, 150, and 180 minutes after consumption. This improvement should give a better indication of any changes in insulin resistance caused by treatments over the course of the study.

Sucralose Study:
Some artificial sweeteners have been shown to alter insulin response, one’s overall wellbeing, and one’s likeliness and willingness to consume food. Artificial sweeteners are important in full-feeding human research studies, however, because of their ability to add variety and a sense of freedom for participants. A relatively new artificial sweetener, sucralose, has not yet been demonstrated to have some of the negative effects other sweeteners have, though extensive independent research has not been done. Before this sweetener should be used in a full feeding study, the effects of sucralose on markers of insulin resistance, concentrations of hunger hormones, and psychological markers of well being including hunger, need to be examined. A brief study was designed to examine these effects by having two subjects in each of four groups consume one treatment drink on each of four days, with eight subjects total consuming each of the four treatments. The treatments were water, water with sucralose, water with sucrose, and water with both sucrose and sucralose. The results thus far have indicated that participants responded to sucralose in a similar manner as they did just water, while they responded to both sucralose and sucrose in a manner similar to sucrose. This indicates that sucralose should be safe to use in a full feeding study without concerns of its effects on the OGTT or on food consumption.

Besides confirming that sucralose is safe to use in the CLA feeding trial, the sucralose study provides several other benefits. The study was designed to take multiple blood samples from intravenous catheters in a fashion that is similar to the protocol to be used in spring 2007. The procedures used during the sucralose study allowed for a more realistic calculation of labor and equipment needs for the CLA study. Also, the sucralose study gave the opportunity to preview nurses’ abilities in a research setting, as well as to act as training for them for the CLA study.

Cooking Methods to Optimize CLA Delivered to Participants
Much is known about the optimal laboratory conditions for the detection and quantification of CLA. The optimal settings in which to prepare high CLA foods, though, are not as well known. To determine the optimal cooking conditions, ground beef with varying amounts of lipid content and varying amounts of CLA were prepared in 100g patties that remained fresh, were subjected to a single freeze/thaw cycles, or were subjected to a simulated freezer burn, and cooked by microwave, convection oven, grill, or frying pan. The lipid in the patties was then extracted within 8 hours of cooking by a modified Folch method after the patties had been cooled to 4˚C. Lipids were then esterified using sodium methoxide in methanol, and analyzed by gas chromatography. The data obtained from the gas chromatograph are currently being analyzed.

The results of this experiment will allow for the preparation of the foods in such a manner that the maximum amount of CLA will be delivered to the participants.

General Conclusion
The efforts of the researchers at Iowa State University have been fruitful in obtaining a multitude of data in a relatively short period of time. These data are being analyzed in a chronology that allows for each step of the research to be completed successfully and accurately. Furthermore, difficulties met thus far have allowed for the optimization of the study that is to be conducted spring 2007. With the experience gained and the data generated, the spring 2007 study successfully produce accurate results in a timely fashion.

Impacts and Contributions/Outcomes

This project will contribute to all of NCR-SARE’s broad-based outcomes and impact sustainable agriculture in the North Central Region. The NCR-SARE’s program benefits the environment by supporting research that provides information to promote sustainable grazing systems that result in soil conservation, improved water quality, and healthier landscapes. The project will add value to milk and beef products raised through pasture-based system and increase market share to increase farmer/rancher profitability.

This project strengthens rural communities by providing options for small to medium dairy and livestock production systems to stay in business by increasing profitability. Each 400-cow dairy operation in northeast Iowa is estimated by Iowa State University to bring in over 16 jobs and nearly $600,000 to local communities each year. The sustainability of these dairies, as well as cattle operations, is dependent on the value of the product they produce. This potential increased value makes this project important to the viability of the rural communities by sustaining the small and medium production systems that provide substantial cash flow.

This project also will impact sustainable agriculture in the North Central Region by providing documentation and education that will increase the demand for CLA-enriched food products that are derived from pasture management systems.

Collaborators: