How Does the Supplementation of Conjugated Linoleic Acid (CLA) affect Human Fat Mass?

Abstract
Research Question: How does the supplementation of conjugated linoleic acid (CLA) affect human body composition? Literature Review: Conjugated linoleic acid (CLA) refers to a family of geometrical and positional isomers of linoleic acid. The predominant sources of CLA are from ruminant animal fats (cow, dairy, sheep, goat, and deer). Over the past 20 years, CLA supplementation has produced positive effects in animal studies, such as significant body fat reduction, anticarcinogenisis, antiatherogenesis, immune modulation and improved bone health. Conversely, human studies have not produced all of the aforementioned results. Nonetheless, there have been many human studies supporting a minimal but significant body fat reduction. These results have been mainly attained with a 50:50 mixture of cis-9, trans-11 and trans-10, cis-12 of purified CLA isomers at various doses. There is evidence indicating that a dose of 3.0g CLA/day may lead to fat loss, whereas the average Canadian consumes between 0.1-1.5 g CLA/day. Therefore, a supplement may be warranted in the future, once further research has been completed on the efficacy and safety of CLA supplementation. Implications to dietetic practice: Clinical dietitians involved in research should experiment with various doses and isomers, but they should particularly investigate the long-term implications of such supplementation. Community dietitians should teach community members how to select natural health products, avoiding inconclusive products like CLA. Administrative dietitians should continue to plan menus based on Canada’s Food Guide, while ignoring CLA claims. Recommendation: In terms of policy, natural health product regulations should be revised, where products sold in Canada should first be proven for efficacy and safety. In terms of research, CLA supplementation does have promising results, and further research should be directed at weight or fat regain post-weight loss. Conclusion: There has been strong evidence in animal studies of CLA’s numerous beneficial properties, such as significant body fat reduction. Nonetheless, body fat reduction is significantly less in human studies, and studies are inconsistent.

Introduction
Conjugated Linoleic Acid, Defined
Conjugated Linoleic Acid, also known as CLA, is a family of positional and geographical isomers of linoleic acid, an omega-6 essential fatty acid. There are four different types of isomers, where the two conjugated bonds can either be cis-cis, trans-trans, cis-trans, or trans-cis. It is important to note that the trans bonds in CLA are not the same as the trans bonds associated with the increased cardiovascular risks caused by decreased HDL-cholesterol, and increased LDL-cholesterol (1, 2).
Biologically, the CLA isomers are produced through the digestive processes of ruminant animals, such as cows, goat’s sheep and deer. In their rumen, the dietary linoleic acid from their feed becomes isomerized by micro bacteria. The first and most abundant metabolic product is termed rumenic acid (RA), which is also known as the cis-9, trans-11, CLA isomer, and consists of 75% - 90% of all naturally produced CLA (Fig.1). A more minor, but important, component of the naturally-produced CLA is the trans-10, cis-12 isomer (Fig.1). Commercially, these isomers are produced from linoleic acid, by heating vegetable oils in the presence of an alkali or by partially hydrogenating the oil (2).
The two isomers found to have biological activity are the cis-9, trans-11 isomer and the trans-10, cis-12 isomer. The last 20 years of research has focused on these two isomers of CLA, and researchers have found some of the defining properties for each isomer. For instance, the trans-10, cis-12 isomer has been identified as the one affecting lipid metabolism and resulting with fat loss. Nonetheless, research using a pure supplement of this isomer has been found to decrease insulin sensitivity in humans and animals, and is therefore not advised for fat loss (1, 6). On the other hand, the cis-9, trans-11 CLA has been found to modulate growth, and when this isomer is mixed equally with the latter, it has been found to cause fat loss without affecting insulin sensitivity (1, 6).

CLA in the Canadian Diet
In the Canadian diet, biological sources of CLA are from the meat and dairy of ruminant animals, such as beef and cow’s milk. Generally, the higher the fat content of the ruminant animal product, the higher the CLA content (3). For instance, homogenized cow’s milk has 5.5 mgCLA/ g fat, and 2% milk has 4.1 mgCLA/g fat (2). Unfortunately, coupled with this higher intake of CLA is a higher intake saturated fat, which is a significant cardiovascular risk factor.
Published reports indicated that Canadians consume about 0.1-1.5 g CLA/day (3). A recent study conducted among healthy young males in Alberta estimate their intake to be at the lower end of this range, 95 mg/day (3, 4). This estimate is similar to published results among Americans (3). These studies utilized methods of food recall, such as the food frequency questionnaire, and focused on the intake of cis-9, trans-11 CLA. Therefore, the published values are likely to be underestimated, particularly because they do not account of the intake of CLA’s only known precursor, trans vaccenic acid (TVA) (3).
Health Implications
There are diverse health implications which were discovered over the last 20 years, and the majority are beneficial. Both human and animal studies have shown positive effects such as decreased body fat and increased lean muscle mass. Also, there is the immune modulation effect where CLA seems to protect against the catabolism and inflammatory responses released by cytokines (2, 7). Other effects have only been observed in animal studies, such as the antiatherogenic effect, where CLA seems to reduce aortic plaque formation, LDL-cholesterol levels, total cholesterol levels and triglyceride levels (2, 7). An anticarcinogenic effect has been seen with regard to the initiation, development and growth of cancer (2, 7). Furthermore, improved bone health has been observed with the prevention of bone loss caused by the natural aging process (2, 7). The main negative consequence of CLA supplementation has been increased insulin resistance, as previously discussed.
This papers aims to answer the question of one particular health implication: “How does the supplementation of conjugated linoleic acid (CLA) affect human fat mass?” First, there will be an overview of the animal research, then human research, followed by a brief discussion on the discrepancies in research results, and the possible mechanisms for this fat reduction. Finally, implications to dietetic practice will be covered, as well as a couple recommendations.

Animal Studies
Background
Over the last 20 years, there have been numerous experiments involving the supplementation of CLA on animals. In terms of CLA’s effects on body composition, most studies have found a significant body fat reduction, however the particular reduction is species-dependent (1, 5, 7). In the case of rats, there have been studies which did not observe a reduction in body fat, and studies which have observed a reduction of 23% body fat (7). In pigs, there has been a wide range of body fat reduction, from a mere 6% to an impressive 25% (7). Similarly, in hamsters the literature shows a body fat reduction of 9% to 24% (7). The most remarkable body fat decrease has been observed in mice, where reductions by as high as 60% have been confirmed by different studies (7). Below is a study examining the effects of CLA supplementation on the body composition of mice.
Research Study
“Conjugated linoleic acid rapidly reduces body fat content in mice without affecting energy intake” by Delany et al. (5)
This study had two purposes, firstly, it was to determine if one of the tested doses of CLA would reduce body fat composition without reducing dietary intake, in mice. Secondly, the study investigated CLA’s effects over time on the body fat composition of mice.
Methods
This study commenced by implementing a high fat diet for all mice, 10 days before beginning the experiments, with ad libitum access to a pre-weighed portion. The first part of the study, investigating dosage, involved experimenting with 60 four-week old mice. The mice were randomly separated into five different dosage groups with the high fat diet: a control group (0%), 0.25%, 0.50%, 0.75% and 1.0% CLA by weight. The specialized diet food produced by Research Diets (NJ), consisted mainly of cis9, trans11 CLA and trans9, cis11 CLA and trans10, cis12 CLA. The animals were weighed seven days before the start of the experiment, and their weight and food intake were then monitored three times a week. At the 39th day, the animals were killed after a three-hour fast, and their blood, plasma, body fat, speen, liver and other organs were analyzed. For the second part of the study, investigating duration, two groups of 40 six-week old mice were used. In this case, the designated diet was either the control (0%), or 1% CLA. Their body weight and food intake was measured twice a week. Eight mice from the control group and eight mice from the treatment group were killed at week 2, 4, 6, 8, 10 and 12 (5). The CLA composition and method of death followed the same protocols as the first part of the experiment.
Significant Results
Significant results for the first part of the study, investigating dose, found significant relationships between CLA consumption and the reduction of body weight (P<0.05), CLA consumption and duration (P<0.01) and finally CLA consumption, over time, with the reduction of body weight (P<0.01). All treatment groups, but one, found no difference in energy consumption in comparison to the control. There was a significant dose-dependent effect of CLA in terms of body fat stores, particularly with the retroperitoneal stores, at the end of the experiment. In terms of the second part of the study, investigating duration, there was a significant weight reduction by the third week which was maintained throughout the 12 weeks (Fig.2). Interestingly, this significant body weight difference between the control and the treatment group became smaller over time, particularly by the end of the study (Fig.2) (5). As with the dose-dependent study, there was no significant difference between energy intakes of the control group vs. the 1 % CLA treatment (Fig.3) (5). Furthermore, retroperitoneal fat stores were most sensitive to fat reduction. In conclusion, feeding mice a diet supplemented with CLA at 1% or less causes a reduction in body fat accumulation and an increase in lean muscle mass accumulation. Moreover, these results are seen early on in the treatment and persist, in a couple cases it was observed from the 2 week mark.
Strengths and Limitations
The strengths and limitations of this study were that it examined varied doses and varied durations, where this allowed researchers to study these variables at length and gather further evidence on their effects. Furthermore, there was a control group in both parts, and this allowed the researchers to reduce confounding variables. Finally, this study had an adjustment period for the introduction of the high fat diet, which reduced the risk of hyperphagia caused by this novel diet. Also, had the CLA supplementation begun simultaneously as the high-fat diet, this may have lead to a conditioned aversion to the new diet, where the CLA could have caused mild behavioural changes. The limitations of this study were that the experimenters knew which animals belonged to the treatment group and those which did not, and this may result in an inadvertent subjective bias. As well, the purpose of the animal studies is to eventually relate this information to human studies, therefore the physiological differences between mice and humans is a limitation.

Human Studies
Background
In contrast with animal studies, human studies have been much less consistent in terms of CLA supplementation causing a significant body fat reduction. Also, the results have been much less drastic in terms of the percent of body fat which was reduced (6, 7). Furthermore, there have been far fewer studies conducted on humans; however these studies are yielding promising results. Most of the studies are 12 weeks or less, and there is only one long-term study to date, by Gaullier et al, which continued for two years; however, there was no control group after the first year (6, 7). Below is a recent study on CLA supplementation in humans, and the following research focus is a meta-analysis of human trials.
Research Study
“Effects of milk supplementation with CLA (isomers cis-9, trans-11 and trans-10, cis-12) on body composition and metabolic syndrome components” by Laso et al. (6)
The purpose of this study was to examine the effects of milk supplementation with CLA (isomers cis9, trans11 and trans10, cis12) on body composition and metabolic syndrome.
Methods
This study was developed over the course of 12 weeks, and involved 60 healthy men and women with indicators of metabolic syndrome. Subjects were randomised to a daily intake of 500 mls of milk supplemented with either 3 g CLA (using a mixture of cis-9, trans-11 and trans-10, cis-12 isomers) or with a placebo. To measure body composition, the researchers used Dualenergy X-ray absorptiometry (DEXA) at the baseline and at the end of the study (6).
Significant Results
There was a small but significant reduction in body fat, particularly in terms of a trend towards a decreased trunk body fat in the CLA treatment group; although this only occurred once the participants were stratified by BMI. As a result, the researchers discovered that only the overweight participants had a reduced fat mass, however the researchers hypothesized that had the study been longer, they may have seen a change in the obese (class I) group. Interestingly, the results among the overweight participants in this study were extrapolated to the results of the Gaullier et al study, at the 3 month mark (Fig.4). Both studies demonstrate a loss of about 3% body fat after three months of CLA supplementation. With regard to the indicators for metabolic syndrome, CLA supplementation had no significant effect on fasting plasma glucose, plasma triglyceride, total cholesterol, HDL-cholesterol and LDL-cholesterol and waist circumference. Furthermore, there was no change in insulin sensitivity, hepatic function nor renal function. In conclusion, the supplementation of milk with 3 g CLA for a three month period produces a significant reduction in body fat in overweight, but not obese patients. Additionally, there were no significant adverse effects or biological changes during this study (6).
Strengths and Limitations
The strengths and limitations of this study begin with the fact that it was a randomized, double blind, placebo controlled trial. This strategy is to protect the results from allocation bias, experimenter bias and a change in the participant’s regular habits. Another strength is that the researchers used the gold standard to measure body composition: DEXA. This method can detect very small changes in body composition, and where they occur. Furthermore, there were stringent inclusion and exclusion criteria, which reduced possible confounders. For example, participants must have maintained a stable weight in the three months prior to the study, defined as a weight change less than 5%. As well, participants were excluded when their energy intake was altered by more than 10% or when a weight change of over 5% occurred during the study. Additionally, participants were screened to ensure that they were healthy, other than metabolic syndrome, and they were not on any drugs which may lead to confounding results. A last strength would be that the dietitian carefully instructed each participant on how to complete the food frequency questionnaire, which would lead to more accurate data. Conversely, there were a couple limitations in this study, for instance participants were classified in exercise categories. There were participants excluded in this study due to a weight loss of 5% or greater, and exercise may have caused this exclusion. Finally, a small percentage of members in the in the CLA group and a moderate percentage of members in the control group showed slight adverse effects in gastrointestinal symptoms such as abdominal discomfort, laxative effects and flatulence. This may promote non-compliance.
Research Study
“Efficacy of CLA for reducing fat mass: a meta-analysis in humans” by Whigham et al.
The purpose of this study was to investigate the role of isomer, dose, and study duration on the efficacy of CLA as a treatment for improving body composition (7).
Methods
The methods involved selecting studies which were longitudinal, randomized, double blind, placebo-controlled human clinical trials. The methods from each study could vary to a certain extent, and participants could be normal weight, overweight or obese and be of any age. Body composition data must have been obtained from validated techniques, for instance DEXA or bioimpedence analysis (BIA). Finally, the dose and type of CLA must be identified in the study. At this time 18 studies met these criteria (7).
Significant Results
Contrary to the animal research, the results from three human studies could not confirm that the trans-10, cis-12 is the CLA isomer responsible for fat loss. In terms of investigating a dose-effect, data from all the studies were plotted comparing the change in fat mass with regard to the dose of CLA given (Fig.5). The doses were from 1 g/day CLA to 6.8 g/day CLA, and the researchers discovered that there was indeed a dose effect, as the regression coefficient was significant. Nevertheless, the dose effect is only significant in comparison with the placebo group, not when compared with the CLA group alone. In terms of investigating a duration-effect, data from all the studies were plotted comparing the change in fat mass over time (Fig.6). The relationship is relatively linear with regard to fat loss during the first six months, and then the relationship reaches an asymptote nearing the second year of treatment. The fat loss over time, in comparison with the placebo group, was calculated to be 0.09 ±0.08 kg/wk. with a dose of 3.2 g/day (7). A couple studies included examined the whether CLA supplementation could inhibit or lessen fat regain after a weight loss period, however the results were insignificant. In conclusion, the body of evidence indicates that CLA supplementation does result in a positive effect on human body composition, namely in inducing a modest fat loss, without altering diet or exercise.
Strengths and Limitations
This meta-analysis included studies which were randomized, double blind, placebo-controlled trials, and as previously discussed, this is the gold standard for research. Also, they chose to only select studies which used validated methods of calculating body composition. Both these criteria were to increase the accuracy and strength of the raw data. Finally, a limitation of this study was the limited amount of studies to select from, as research in human trials has only begun. Once more studies are available, a more complete meta-analysis can be conducted.
Why the discrepancies?
There are numerous discrepancies among animal research, human research and between these two domains of CLA investigation. Firstly, there is a lack of reproducibility in animal trials compared to clinical trials, and this can be due to the variance in the age of subject and/or the their developmental stage, gender and the genetics of the subject in terms of their predisposition for fat accumulation and storage (8). Furthermore, many clinical trials did not control for energy intake or energy expenditure through exercise, which are confounders in terms of fat metabolism and reduction (2). Finally, the type of isomer or isomer mix, dose and duration has varied greatly between trials, making it difficult to draw conclusions. Comparing the dose given to a mouse with the dose given to a human, it can be viewed as similar or completely different. In terms of a dose relative to body weight, the dose is much higher for mice throughout the research. However if you compared the dose as a percentage of their daily calories, the dose is similar for humans as it is for mice (7). Finally, CLA’s metabolism in the body is unclear, as are its mechanisms for fat reduction. Different possibilities found in the research thus far include adipocyte apoptosis, the reduction in accumulation of fatty acids and / or triglycerides in adipocytes and the increase in metabolic rate. Other possibilities include the down-regulation of leptin expression, the reduction in food intake and the increase in fatty acid oxidation (7, 8).
Implications to dietetic practice
Clinical dietitians involved in research should experiment with various doses and isomers, but they should particularly investigate the long-term implications of such supplementation. In a community setting, dietitians should teach members of the community how to select natural health products and avoid inconclusive products such as CLA. Finally, administrative dietitians should continue to plan menus based on Canada’s Food Guide, while ignoring CLA claims. If adequate evidence does arise, dietitians involved with the food industry should encourage a change in breeding methods to increase the natural production of CLA. A recommendation would be that the natural health product regulations should be revised, where products sold in Canada should first be proven for safety, but also efficacy. In terms of research, CLA supplementation does have promising results, and further research should be directed at the fat regain during post-weight loss periods, and whether CLA can reduce this regain.
Conclusion
There has been strong evidence in animal studies of CLA’s numerous beneficial properties, such as significant body fat reduction. Nonetheless, body fat reduction is significantly less in human studies, and studies are inconsistent. The recent meta-analysis on human trials calculated a fat loss of 0.09 kg/week resulting from CLA supplementation of a mean dose of 3.2 g/day (7). This fat loss is modest; however it is important in view that Americans have the tendency to gain an average of 0.009 kg/week (7), and Canadians are following the same trend of weight gain. The public should be discouraged to consume these supplements at this time, as the evidence is insufficient to recommend a specific dose, the isomer mix and the duration for supplementation. Furthermore, a dose composed of an equal blend of cis-9, trans-11 and trans-10, cis-12 CLA isomers has been proven safe for short periods of supplementation, however there is still research needed to prove its long-term safety.

References
1. Wang, YW and Jones, PJH. Conjugated Linoleic Acid and Obesity Control: Efficacy and Mechanisms. International Journal of Obesity, 2004; 28, 941-955
2. Rainer, L et al. Conjugated Linoleic Acid: Health Implications and Effects on Body Composition. American Dietetic Association, 2004; 104:963-968
3. Ens JG et al. An assessment of c9t11 linoleic acid intake in a small group of young Canadians. Nutr Res. 2001; 21:955-60
4. Ritzenthaler KL, et al. Estimation of conjugated linoleic acid intake by written dietary assessment methodologies underestimates actual intake evaluated by food duplicate methodology. J Nutr. 2001; 131:1548-5
5. Delany et al. CLA rapidly reduces body fat content in mice without affecting energy intake. American Journal of Physiology, 1999; 276:R1172-R1179
6. Laso et al. Effects of milk supplementation with CLA (isomers cis-9, trans-11 and trans-10, cis-12) on body composition and metabolic syndrome components. British Journal of Nutrition, 2007; 98, 860-867
7. Whigham et al. Efficacy of CLA for reducing fat mass: a meta-analysis in humans. American Journal of Clinical Nutrition, 2007; 85: 1203-11
8. Plourde, M et al. Conjugated Linoleic Acid: why the Discrepancy between Animal and Human Studies? International Life Sciences Institute, 2008; 66(7); 415-421

Appendix

Fig.1: Linoleic acid and its derivative CLA isomers, trans-10, cis-12 and cis-9, trans-11

Fig. 2. Body weights of control and 1.0% CLA treatment group upon death at 2, 4, 6, 8, and 12 wk after beginning the CLA diet. *P <0.005, difference from control.

Fig. 3. Cumulative energy intake for control group and 1% CLA groups fed CLA for 2, 4, 6, 8, or 12 wk. Each bar represents the mean 6 SE.

Fig.4: Figure taken from Gaullier et al. and modified for the present study. BFM is body fat mass.

Fig.5: The effect of CLA dose on rate of change in fat mass, relative to the placebo. The circled data point is an outlier, and was not included in regression analysis.

Fig.6: Change in fat mass over the course of the CLA treatment