Progress 10/01/06 to 09/30/10
Outputs
OUTPUTS: During the course of this study, three scientific papers were published, one student received his Ph.D. (Shane M. Huebner, currently post-doctoral fellow with Professor Susan Smith, Nutritional Sciences, University of Wisconsin), an undergraduate student (James P. Campbell, currently in MD/PhD studies at Medical School of Wisconsin, Milwaukee) completely his senior thesis using funds from a Hilldale and Cargill/Benevenga Scholarship Grant. During the course of this study, a NMR metabolome for arthritis was developed. Data demonstrating the anti-inflammatory effects of conjugated linoleic acid has been shared with a manufacturer of dietary conjugated linoleic acid supplements as well as two global pharmaceutical companies. However to date, the use or the value of conjugated linoleic acid as a useful supplement or food additive to control inflammatory disease is not well recognized by either the scientific community or the general public. Data have been collected for the publication of at least four additional peer reviewed scientific publications (see dissertation in publication list below). PARTICIPANTS: Professor Mark E. Cook, in the Animal Sciences Department at the University of Wisconsin-Madison, served as the principal investigator on the project. Shane M. Huebner was the lead graduate student who conducted this work as part of his doctoral research. Dr. Huebner received his doctorate in philosophy through the Interdepartmental Graduate Program in Nutrition Sciences at University of Wisconsin-Madison in December, 2010, and currently is a postdoctoral fellow with Professor Susan Smith in Nutritional Sciences at University of Wisconsin-Madison. During his graduate studies, Dr. Huebner mentored three undergraduate student. One of these students, James P. Campbell,received both a Cargill/Benevenga Scholarship and a Hilldale Scholarship at the University of Wisconsin to conduct independent studies on conjugated linoleic acid and arthritis. Mr. Campbell, upon completing his undergraduate degree in Nutritional Sciences, entered a fully paid Medical School Doctoral/Doctor of Philosophy study at the Medical College of Wisconsin-Milwaukee. Katherine E. Pankratz also received both the Cargill/Benevenga and Hilldale Scholarships to conduct her senior honors thesis on environmental factors that influence the severity of inflammation. Ms. Pankratz is currently a Veterinary student at the University of Wisconsin. A high school student, Tyler G. Fulmer, who was part of the Youth Apprentice Program-Biotechnology (now an undergraduate student), continues to work in the Cook lab. Dr. Daniel E. Butz, Assistant Scientist was instrumental in training Dr. Huebner on procedures for studying arthritis. Dr. Butz played a critical role in the development of the arthritic metabolome along with Dr. Fariba Assadi-Porter (Biochemistry). Dr. Butz continues 60% at the University of Wisconsin, and serves part-time as Chief Scientific Officer at Isomark (a company that has a technology that detects the onset of infection using breath). Dr. Annette Gendron-Fitzpatrick in Research Animal Resource Center conducted is histological analysis of tissues. TARGET AUDIENCES: There are three primary target audiences. The first target audience consists of the general public. The public is becoming increasingly interested in diet and the control of chronic inflammatory diseases, like arthritis. At this point, little attempt has been made to reach this audience. The second target audience is producers of foods that are either naturally enriched in conjugated linoleic acid (CLA), such as dairy products or meat from ruminant animals or products where CLA can be added. At this point, not enough scientific literature has been published on CLA and inflammation to provide these producer groups with a good science basis to market a CLA product for controlling inflammation. The third target audience is the scientific community. During this project, results were shared in the form of abstracts at scientific meetings. The paper published in Journal of Nutrition in 2010 is the first step in getting new information into the hands of scientists. Expected are at least 5 additional papers within the next 18 months. Once these works are published, the studies on CLA and arthritis will go through an important period of critical evaluation and if found to have sound scientific merit, the information will begin moving to the other target audiences. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
During the four years of investigating the effects of conjugated linoleic acid (CLA) on arthritis, a total of six studies were completed. In the first study, it was observed that mixed isomers of CLA (c9t11- and t10c12-CLA, in a 50:50 ratio) reduced inflammation in an arthritis model (collagen-antibody-induced arthritis) that was independent of an adaptive immune response. Feeding CLA (mixed isomers) prior to the induction of arthritis by immunization with type II collagen (known as collagen-induced arthritis, or CIA) tended to result in signs of inflammation earlier, but the severity of inflammation later in life appeared to be attenuated. Research was redirected to study CLA's anti-inflammatory effects after arthritis had already developed. Studies also were conducted to determine which CLA isomer was an anti-inflammatory agent, what was the minimal level of the active isomer needed to have anti-inflammatory effects, and to determine if natural sources of CLA (c9t11-CLA in butter) was anti-inflammatory in the CIA model. It was found that both isomers of CLA (c9t11- and t10c12-CLA) possessed anti-inflammatory properties. The t10c12-CLA isomer was found to have greater anti-inflammatory activity than the c9t11-CLA isomer. As the dietary dose of t10c12-CLA increased in the diet from 0 to 0.5%, the severity of arthritis decreased in a dose dependent manner (higher doses may have had greater anti-inflammatory activity). The c9t11-CLA isomer reduced inflammation when as little as 0.125% was added to the diet. Increasing the dietary levels above 0.125% (e.g., to 0.5%) did not result in further reductions in arthritic signs. Butter containing 0.07% c9t11-CLA and its precursor 18:1, t11 resulted in levels of hepatic CLA (c9t11-CLA) not different from 0.125% synthetic dietary CLA (c9t11-CLA), and the anti-inflammatory affects of butter were equal to 0.125% synthetic CLA in the diet. Enriching butter with higher levels of c9t11-CLA and 18:1, t11 did not result in further decreases in arthritic scores. Also found was that the t10c12-CLA was an effective anti-inflammatory independent of the severity of arthritis. Both isomers of CLA were effective at preventing arthritis-induced increases in plasma tumor necrosis factor alpha and interleukin-1. Neither isomer affected anti-collagen antibody responses. Arthritis-induced hypoaminoacidemia was prevented by feeding CLA, but the t10c12-CLA isomer appeared more effective than the c9t11-CLA isomer. Hatch provided a significant amount of the funds to cover labor (graduate student) associated with this project. Additional funding was supplied using royalty income and additional small grants for training undergraduate students (Cargill/Benevenga, and Hilldale Scholarships). Preliminary trials on metabolomics that resulted in one of the studies provided critical preliminary data to obtain more funding and to expand the study of CLA's anti-inflammatory actions.
Publications
- Huebner, S.M. 2010. Individual isomers (cis-9, trans-11 and trans-10, cis-12) of dietary conjugated linoleic acid reduce inflammation in a murine collagen induced arthritis model. Dissertation, University of Wisconsin-Madison.
- Huebner, S.M., Butz, D.E., Campbell, J.P., Cook, M.E., and Assadi-Porter, F.M. 2010.Changes in small molecular weight biomarkers identified by NMR spectrometry in response to dietary treatment with two conjugated linoleic acid isomers (c9,t11; t10c12) in a murine collagen-induced arthritis model. FASEB J. 24:542.4
- Huebner, S.M., Campbell, J.P., Butz, D.E., Fulmer, T.G., Gendron-Fitzpatrick, A., and Cook, M.E. 2010. Individual isomers of conjugated linoleic acid reduce inflammation associated with established collagen-induced arthritis in DBA/1 mice. J. Nutr. 140:1454-1461.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: During the reporting period, 2009, new methods for evaluating the mechanism by which conjugated linoleic acid (CLA) reduces inflammation were developed. Using Nuclear Magnetic Resonance Spectroscopy (NMR) a metabolome of small molecules in the plasma of mice with arthritis when fed a control or CLA supplemented diet was constructed. We also developed cytokine and antibody assay systems to determine the effects of dietary CLA on key initiators of inflammation. To increase support and expand research on the role of CLA on chronic inflammation, a pre-proposal was submitted to the National Dairy Council (NDC) to study the effects of the c9, t11-CLA isomer, found in butter, on the arthritic inflammatory metabolome in mice. NDC requested a full proposal, which was submitted, but not funded. An abstract was published and presented at the Experimental Biology meetings in New Orleans, LA. An undergraduate student, James Campbell, who has been assisting on the project, submitted a grant proposal, and received a Hilldale Scholarship to study CLA and arthritis. The first paper involving this Hatch proposal was submitted. Also during the reporting, previous work had suggested that the effects of dietary CLA on inflammation in the model used, may be dependent on the severity of inflammation and the type of basal diet used. An experiment was conducted to investigate basal diet (casein versus soybean protein based) and level of co-stimulation with Mycobacterium tuberculosis. PARTICIPANTS: Shane Huebner was the graduate student supported on this project. During the project, Huebner mentored several undergraduate students: James Campbell, Katherine Pankratz, and Tyler Fulmer. Both Campbell and Pankratz received additional support to work on experiments related to this project (both received a Cargill/Benevenga Research Scholarship, and Hilldale Research Scholarship). Fulmer began work with Huebner as part of a High School apprenticeship program known as the Biotechnology Youth Apprentice, sponsored by Promega. Upon completion of the program, Fulmer remained as a part time student worker in the laboratory. Drs. Dan E. Butz (Scientist in Zoology/Animal Sciences), Fariba Assadi-Porter (Scientist in Biochemistry), and Hamid Eghabalnia (Professor, Univ. Cinn.) participated as collaborators on this research project and were specifically involved in the development of the arthritic metabolome. TARGET AUDIENCES: The first public release of information made related to this project was to fellow scientists at the Experimental Biology meeting in New Orleans, LA (April, 2009). A paper has been submitted for publication that shows the effectiveness of both the c9, t11- and t10, c12-CLA isomers at reducing arthritic inflammation in the mouse model. The PI presented a paper entitled "Vaccination taxation" at the 5th International Veterinary Vaccine and Diagnostic Conference, Madison, WI, July 19, 2009, that reported on the anti-inflammatory effects of CLA. PROJECT MODIFICATIONS: A major goal of this project was to also investigate the effectiveness of CLA isomers in the matrix of milk fat. Milk enriched in c9, t11- and t10, c12-CLA was produced, but to date has not been specifically studied in the arthritic model. Initially, the finding that the t10, c12-CLA isomer was the most effective isomer at reducing arthritis resulted in increased attention on this isomer. We were unsuccessful in generating enough t10, c12-CLA in milk fat to create a reasonable hypothesis that milk fat could reduce inflammation. However, more recent findings that the c9, t11-CLA isomer was effective in reducing inflammation in the arthritis model (involving recent improvements in the model) has renewed interest in the study of milk fat. It is hoped that an experiment will be conducted during the final year of the project to investigate milk fat in arthritis. In order to accomplish this final goal, we will have to obtain additional support. Three proposals have been written to support this objective, but to date, none of these proposals have been supported, even though the review report have been largely supportive. The use of NMR analysis of plasma of samples collected from the studies conducted to date has opened new opportunities for finding support for this project.
Impacts
In our initial experiments, it was found that the anti-inflammatory effects of conjugated linoleic acid (CLA) was due to the t10, c12 isomer (t10, c12-CLA). However, newer findings clearly demonstrated that the c9, t11-CLA isomer was also effective at reducing arthritic inflammation. The t10, c12-CLA isomer is a minor isomer in food products from ruminant animals (e.g., milk and meat), whereas the c9, t11-CLA isomer can represent as much as 5% of the fatty acids in fats from ruminant sources. In addition, vaccenic acid (C18:1, t11) can be as much as 6% of the ruminant fatty acids and can be converted by hepatic desaturase to c9, t11-CLA. The finding that c9, t11-CLA reduces inflammation supported a conclusion that fat from ruminant products have anti-inflammatory activity. The consumption of fats from ruminant origin has been historically considered "unhealthy." Findings that fatty acids in ruminant fats actually protect against inflammatory disease could counteract the arguments against eating dairy products or the consumption of animal fats. Recently, animal scientists have found new feeding strategies that will elevate the content of CLAs in animal fat. Implementation of new feeding strategies that increased CLAs in animal fats may provide food producers new marketing strategies for products of ruminant origin.
Publications
- Huebner, S.M., J. Campbell, M. Loy, D.E. Butz, and M.E. Cook. 2009. Conjugated linoleic acid reduces joint inflammation in a murine collagen-induced arthritis model FASEB J. 23:909.4.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: During the reporting period, current results of the anti-inflammatory properties of conjugated linoleic acid (CLA) in a murine model of collagen-induced arthritis have been disseminated to interested parties of the work (Cognis, Inc. & Wisconsin Alumni Research Foundation). Interest in the results of this grant has increased recently due to the approval of conjugated linoleic acid (CLA) for use in both animal and human foods by the Food and Drug Administration. Additionally, work from this grant has led to new research opportunities. A pre-proposal was submitted to the National Dairy Council investigating the anti-inflammatory attributes of dietary casein but unfortunately was not funded. A graduate student became a mentor to an undergraduate student for a Biology 152 project based upon work associated with this grant. Additionally, this undergraduate student has received funding through the Cargill grant to continue additional research opportunities associated with this project. Finally, an abstract titled "Conjugated linoleic acid reduces joint inflammation associated with collagen-induced arthritis in mice" has been submitted to the Experimental Biology 2009 conference summarizing current work completed related to this grant. PARTICIPANTS: Individuals at the University of Wisconsin that worked on the project included Shane Huebner (lead funded project graduate student with PI), David Trott (graduate student with PI), James Campbell (undergraduate mentored by Huebner), Niels Jorgensen (undergraduate summer student worker from Lawrence University, Appleton, WI), Tyler Fulmer (Promega High School Youth Apprentice mentored by Huebner), and Dan Butz (former graduate student of PI and currently Assistant Scientist in Zoology). Both Campbell and Fuller are mentored students on the project. TARGET AUDIENCES: At the time of this report, the key target audiences have not been made aware of the results. When final papers are published, the key audiences will be producers and marketers of products and supplements containing conjugated linoleic acid (CLA), and food producers adding CLA to select foods. The general public is anticipated to be an audience interested in the findings. Food choices by the public could be influence by the findings. PROJECT MODIFICATIONS: There are no anticipated major changes to the project. Experimental design modifications will include determining the minimal level of each isomer of CLA that has anti-inflammatory effects. This finding should be critical in the estimate of the anti-inflammatory effects of CLA found in food products and supplements.
Impacts
Current work has confirmed that CLA is effective in reducing inflammation associated with collagen-induced arthritis in mice. Additionally, the t10,c12 CLA isomer fed at 0.5% diet by weight has been shown to be the most effective in reducing this inflammation. Since the t10,c12 CLA isomer has been shown to be very difficult to enrich in milk fat (previous difficulties documented in earlier progress report), a dose study must be conducted investigating if lower concentrations of t10,c12 CLA, similar to concentrations that can be enriched in milk fat (~0.03-0.05%), can be effective in reducing inflammation. Conversely, the c9,t11 CLA isomer fed at 0.5% diet by weight has also been shown to reduce inflammation associated with arthritis. Since this isomer can be enriched in milk fat at levels approaching 5% or higher, more investigation is warranted studying c9,t11 CLA isomer's role in reducing inflammation. If either isomer of CLA can be enriched in milk fat at a percentage determined to be anti-inflammatory, commercial production of dairy foods and dietary recommendations would be impacted to take advantage of CLA nutritional and anti-inflammatory properties. Additionally, current work has led to a new collaboration with Dr. Fariba Assadi-Porter (Assistant Scientist, Biochemistry, University of Wisconsin-Madison) investigating the role of CLA's effects upon the metabolome and its relationship with chronic inflammation.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Previous work was completed and disseminated in a published referred journal article that determined the effects of synthetic mixed isomers of conjugated linoleic acid (CLA) on inflammation associated with both the collagen-induced and collagen antibody-induced models of arthritis. This report showed that dietary levels of the mixed isomers of CLA reduced inflammation, however the reduction in inflammation depended on the model used and the timing of CLA feeding relative to the induction of arthritis. In another objective of this work, the effects of CLA, naturally found in foods, on inflammation was begun. To develop CLA enriched fats, dairy cattle diets were formulated (based on previous work by others) and fed to lactating cows to generate butter fat enriched in either the c9,t11-CLA or the t10,c12-CLA isomer. Milk from cows fed the specific CLA-isomer enhancing diet was collected over 3 days at time points previously shown to result in elevated levels of each CLA isomer.
Collected milk was separated into cream and then converted into butter. Approximately 1 Kg of 3 types of butter was made (control, and butter enriched with either 5% c9,t11-CLA or .03% t10,c12-CLA, as expressed as a percent of total fatty acids). These enriched butter lots will be used in future experiments studying the effects of natural sources of CLA on inflammation. Experiments have been conducted, and are still ongoing, to examine the anti-inflammatory effects of the individual CLA isomers using pure isomer sources.
PARTICIPANTS: Shane Huebner, Nutritional Science graduate student coordinated and conducted trials investigating the effects of individual CLA isomers on collagen-induced arthritis. Huebner also coordinated and conducted the dairy cattle feeding and milk collection trial. Professor Dave Combs (UW-Madison, Dairy Science Department) assisted in the formulation of the dairy cattle diets. Staff at the Dairy Cattle Center at UW-Madison and the Arlington Agriculture Research Station assisted in the dairy cattle feeding study. Lee Jensen (Laboratory Manager in the Department of Food Science, UW-Madison) provided direction and assistance in the conversion of the milk fat to butter. James Campbell and Meaghan Loy, undergraduate students at UW, assisted Huebner in the rodent studies.
TARGET AUDIENCES: At this time, the appropriate audience for this report are scientist investigating the effects of CLA on inflammation.
PROJECT MODIFICATIONS: In the original proposal, we planned to investigate the effects of naturally occurring CLA on acute inflammation using the rodent endotoxin response. We now plan to use a rodent model that is more relevant to septic shock following surgery. The model that will be used mimics bowel perforation and a resulting peritonitis. Also, at this time, we have not been able to achieve a level of t10,c12-CLA in butter that has been previously reported. Hence, our initial work on naturally occurring CLA in milk fat will focus on the main isomer found, c9,t11-CLA. The c9,t11-CLA isomer has been previously shown to have an effect on the release of the pro-inflammatory cytokine, tumor necrosis factor. Since this is the major naturally occurring CLA isomer in milk and since this isomer is associated with anti-inflammatory effects, we will focus primarily on c9,t11-CLA's role in inflammation at this time.
Impacts
In our report on 2006 (see Butz et al., 2007) we described experiments that demonstrated in mouse models, that dietary mixtures of CLA were effective at reducing inflammation in an arthritis model. While premature, numerous web sites have now reported a possible benefit of CLA or foods enriched in CLA for the treatment of inflammation associated with arthritis. In 2007, experimental data collected suggested that the t10,c12-CLA isomer was the most effective isomer in reducing inflammation in a mouse model of collagen-induced arthritis. The design of these experiments only investigated the effects of the individual isomers when fed post arthritis induction. If these results are confirmed in human clinical trials, the contribution of foods enriched in select CLA isomers could be evaluated for potential functional effects. Since the t10,c12 isomer of CLA is a minor CLA isomer, even when enriched in animal fats through natural feed ingredient formulation, the contribution
of this CLA isomer to the anti-inflammatory effects of select animal fats would be questionable. For example, while we were able to increase the c9,t11-CLA content of butter to 5% of the total fatty acids, using feeding formulation previously suggested to increase t10,c12-CLA, the c10,t12-CLA isomer was increased to only resulted .03% of the fatty acids. This level of t10,c12-CLA in animal fats is unlikely to be adequate to have the anti-inflammatory effects found with feeding the pure isomer at .25% of the total animal diet. The over interpretation of an experimental result, even those described above, could represent a premature dietary recommendation affecting human health. However, in our previous studies we have found that the c9,t11-CLA isomer has an ability to reduce the release of the inflammatory cytokine, tumor necrosis factor. Additional studies using the arthritis model may demonstrate methods in which c9,t11-CLA could be useful in specific areas of inflammatory disease.
Publications
- Butz DE, Li G, Huebner SM, Cook ME. 2007. A mechanistic approach to understanding conjugated linoleic acid's role in inflammation using murine models of rheumatoid arthritis. Am J Physiol Regul Integr Comp Physiol. 2007 Aug;293(2):R669-76.
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Progress 01/01/06 to 12/31/06
Outputs
Preliminary trials that led to the funding of this work were completed and published or submitted for publication. It was found that the t10, c12 isomer of conjugated linoleic acid (CLA) resulted in compensatory gain following endotoxin-induced weight loss. A mixture of c9, t11 and t10, c12 isomers of CLA reduced the amount of weight loss caused by endotoxin injection. Dietary t10, c12 isomer of CLA was found to reduce plasma tumor necrosis factor (TNF) 2 hr after endotoxin injection. A dietary mixture of the t10, c12 and t9, c11 isomers of CLA were effective at reducing collagen antibody-induced arthritis and plasma TNF in mice. Collagen-induced arthritis appeared earlier if CLA was fed prior to collagen injection or later when fed at the same time as injection when compared to control fed mice. Peak arthritis scores were not affected by CLA feeding the first 80 days. Relapse of inflammation (after day 80) was reduced in mice fed both isomers compared to control fed
mice. Using these models, butter enriched with individual isomers of CLA will be used to assess the role of milk fat on inflammation and weight loss in the two models of arthritis. Plans are underway to feed cows diets which will increase select isomers of CLA in milk fat. The different milk fats will be studied to assess the role of milk fat composition on anti-inflammatory affects.
Impacts
Previous work has shown that dietary CLA has anti-inflammatory effects in models of inflammation involving airway, gastrointestinal tract, kidney (Lupus), and cancer or endotoxin-induced cachexia. While dietary supplements are available for human use of CLA, little is known about the effects of naturally occurring CLA in human diets. The primary source of CLA in human diets is the fat from ruminate animals. Milk fat CLA content can be altered through the method of feeding ruminants (for example, cows that are fed on pasture have as much as a 3 fold increase in CLA content). The goal of this research will be determine if milk fat enriched with CLA through natural feeding systems will show anti-inflammatory actions using a model of arthritis in mice. These findings should provide insight into a health benefit associated from the consumption of animal fat.
Publications
- Butz, D.E., G. Li, M.E. Cook. 2006. t10, c12 conjugated linoleic acid induces compensatory growth after immune-challenge. J. Nutr. Biochem. 17:735-741.
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