DFA: Conjugated Linoleic Acid (CLA) Supplementation and Intermediate Health Outcomes (2011)

Study Design:
- Click here for explanation of classification scheme.
Quality Rating:
Research Purpose:
  • To determine whether 14-week CLA supplementation as triacylglycerols (3.76g) with a 50:50 combination of the two main isomers (cis9,trans-11 and trans-10,cis-12) added to flavored yogurt-like products was able to alter body composition in healthy human adults
  • In addition, subcutaneous abdominal adipose tissue biopsies were performed to investigate in vivo the changes in the expression of several key adipose tissue genes, including PPAR gamma, lipoprotein lipase (LPL), UCP-2 and hormone-sensitive lipase (HSL).
Inclusion Criteria:
  • BMI 23 to 27.5kg/m2 with a stable body weight for at least three months
  • At least 135 minutes of moderate exercise per week
  • Fasting plasma cholesterol less than 6.5mmol per L
  • Fasting plasma triacylglycerol less than 2mmol per L
  • Fasting plasma glucose less than 7mmol per L
  • Not regular consumers of any fatty acid nutritional supplements or drugs known to affect body weight, lipid or glucose metabolism except for oral contraceptives for women.
Exclusion Criteria:

Pregnant women.

Description of Study Protocol:


Recruited from the local community by advertisement.


  • Randomized double-blind placebo-controlled
  • Method of randomization was not given.

Blinding Used

Double blind.


  • Placebo
  • CLA

Statistical Analysis

  • Differences between groups from baseline to day 98 were assessed using overall tests
  • In the case of a significant overall test result, a Wilcoxon non-parametric test was used for differences between groups
  • An interaction test was used to determine the effects of CLA, sex, type of subject (fat or lean) and the interactions CLA x sex and CLA x type of subject on body composition
  • According to prior work (Blankson et al, 2000), it was estimated that 20 subjects per group would be needed to detect a 5% change in body fat mass levels at a significant level of 0.05 using a power of 0.85 and a standard deviation of six.
Data Collection Summary:

Timing of Measurements

  • The duration of the study was 14 weeks (98 days)
  • Subjects had blood sampling at 7:30 A.M. following a 12-hour overnight fast on days zero, 42 and 98. Body composition (days zero and 98) and other laboratory analyses were also performed on these visits.
  • On days zero and 98, subcutaneous abdominal adipose tissue biopsies were performed.

Dependent Variables

  • Body composition: After urine collection, weight and height were measured. Total lean body mass and total fat mass were measured using a dual-energy X-ray absorptiometer (Hologic QDR 4500 A).
  • Blood analyses: Glycemia, insulinemia, triacylglycerols, NEFA, total and HDL-cholesterol, liver enzymes, blood cell count, bleeding time, prothrombin, activated cephalin time, thiobarbituric acid reactive substances, CLA and fatty acid profile. Plasma fatty acids and CLA isomer concentrations were measured by GC-MS.
  • Abdominal adipose tissue steady-state expression level of mRNA encoding HSL, LPL, UCP-2 and PPAR-gamma: Biopsies were performed under local anesthesia by aspiration from the per-umbilical area through a 15-gauge needle. The concentrations of the target mRNA were measured by RT-competitive PCR.
  • Respiratory exchange measurements were performed during a one-hour period by indirect calorimetry (Delta-trac Metabolic Monitor). For nitrogen excretion rate, urine was collected during the calorimetry measurements for measurement of this. Total lipid oxidation rate and RMR were calculated.

Independent Variables

  • The yogurt-like products were made using a standard dairy process for fermented milk using skimmed milk, 8% sugar, cherry flavor and 3.76g triacylglycerols TONALIN 75 TG for the CLA group and cream for the control group by Danone Company
  • Placebo: Yogurt only
  • CLA: Mixture contained 35% cis-9,trans-11 isomer and 35% trans-10,cis-12 isomer. The major remaining fatty acids were 8.3% C16-0, 2.9% C18-0, 14.8% 18:1n-9 and 1.4% 18:2n-6. Less than 1% of trans-trans CLA isomers were found. This composition was verified by GC-MS.
Description of Actual Data Sample:
  • Initial N: 44 (22 males, 22 females)
  • Attrition (final N): 44; biopsies were taken from only 22 subjects [11 subjects in the placebo group (six males, five females) and 11 subjects in the CLA group (five males, six females)]
  • Age:
    • Placebo: 28.5±1.14
    • CLA: 29.4±1.39
  • Anthropometrics: Baseline characteristics were similar in both groups (refer to table in results) and also in the subgroup chosen for biopsy (data was not shown).
Summary of Results:

 Key Findings

  • The consumption of CLA-enriched yogurt-like products for 14 weeks (98 days) affected neither the body weight nor the BMI of either the men or women
  • Fat mass and fat free mass were unaffected by CLA supplementation
  • There was no effect of sex or type of subject (fat or lean) using an interaction test (data was not shown)
  • CLA consumption did not alter glycemia, insulinemia, leptinemia, triacylglycerols, total HDL-cholesterol concentrations and plasma NEFA concentrations
  • Plasma CLA cis-9, trans-11 levels increased with CLA consumption (29.9±3.1 and 57.3±7.2umol per L on day zero and day 98, respectively, P<0.02), whereas concentrations remained stable in the placebo group (37.2±3.6 and 40.8±3.4umol per L on day zero and day 98, respectively)
  • CLA trans-10,cis-12 isomer was undetectable in plasma during the whole study in the placebo group while it increased significantly in the CLA-treated group (undetectable on day zero and 18.2±3.6 on day 98, P<0.01).

Selected Results on Body Composition



Day  Zero (N=23)


Day 98 (N=23)


Day Zero (N=21)


Day 98 (N=21)

BMI (kg/m2)





Fat mass (kg)

19.4±1.2 18.9±1.3  18.2±1.1 18.2±1.2

Free fat mass (kg)

52.9±2.0 53.7±2.1 56.1±2.3 56.7±2.2

Other Findings

  • CLA-enriched products were well tolerated as no adverse events occurred
  • Compliance was 99.9%, using a product count every two weeks on the occasion of each yogurt-like product delivery
  • No changes were observed in blood cell count, liver enzyme levels, bleeding time and pro-thrombin concentration (data was not shown)
  • Activated cephalin time was significantly increased in men from the CLA group (33.6±1.1 and 34.4±1.4s, on day zero and day 98, respectively, P=0.01) but not in women (31.1±0.8s on day zero and day 98, respectively, NS)
  • Thiobarbituric acid reactive substance formation was unaltered by CLA consumption in either men or women
  • CLA did not alter the relative concentration of n-3 or n-6 PUFA in plasma NEFA
  • In adipose tissue, CLA trans-12,cis-10 isomer was undetectable in both groups on day zero and remained undetectable in the placebo group on day 98 but increased significantly in the CLA group from zero to 1.96 ± 0.4nmol per mg. CLA cis-9,trans-11 isomer increased significantly from 8.26±0.5 to 10.47±0.9nmol per mg (P=0.01) in the CLA group between day zero and day 98 but remained stable in the placebo group.
  • Total lipid oxidation rates were affected by CLA consumption (0.68±0.06mg per kg per minute vs. 0.58±0.07mg per kg per minute on day 98 and day zero, respectively)
  • When referred to total body weight, basal energy expenditure tended to increase more in the CLA group (93.2±1.9kJ per kg per day on day 98 vs. 89.5±1.8kJ per kg per day on day zero, P=0.07), whereas no changes were observed in the placebo group
  • When basal energy expenditure was expressed by reference to the kg free fat mass, a significant increase was found in the CLA group (123.3±2.5kJ per kg free fat mass per day on day 98 vs. 118.7±kJ per kg free fat mass per day on day zero, P=0.03). No change was found in the placebo group.
  • PPAR gamma mRNA expression increased significantly in the CLA group (31.8amol per ug total RNA at day zero vs. 44.4amol per ug total RNA at day 98, P<0.01) while it remained unchanged in the placebo group
  • HSL mRNA levels were reduced by CLA (-42%±7%, P=0.01) and were unchanged in the placebo group
  • UCP-2 and LPL mRNAs were unchanged by either CLA or placebo.
Author Conclusion:
  • A 98-day supplementation with a 50:50 mixture of the two CLA isomers cis-9,trans-11 and trans-10,cis-12 in a flavored yogurt-like product was unable to alter body composition, although a significant increase in the RMR could be recorded in the subjects of the CLA group
  • It seems therefore that the 14 weeks of the trial were not long enough to produce significant changes in body composition
  • It is important to note that, to the authors' knowledge, this is the first observation of CLA supplementation-induced gene expression in human tissues.
Funding Source:
Dannon made the yogurts; TONALIN from Natural Lipids Ltd
Food Company:
Pharmaceutical/Dietary Supplement Company:
Other: no information on funding given
In-Kind support reported by Industry: Yes
Reviewer Comments:
  • The study design section does not mention or describe subject type and how subjects were classified as fat or lean, as is mentioned in the statistical analysis section. The BMI range was 23 to 27.5, so this reviewer questions the use of fat vs. lean.
  • The statistical analysis section is not very clear on all of the statistical methods used and this reviewer questions the appropriateness of the statistical analysis
  • If CLA was incorporated into triacylglycerols, then this reviewer questions why triacylglycerols without CLA were not used for the placebo instead of cream. What were the differences between the two?
  • Compliance was measured only using product count. However, they did also use plasma CLA concentrations and did see an increase with CLA supplementation, thus confirming compliance with consumption of the supplement.
Quality Criteria Checklist: Primary Research
Relevance Questions
  1. Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? (Not Applicable for some epidemiological studies) Yes
  2. Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about? Yes
  3. Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dieteticspractice? Yes
  4. Is the intervention or procedure feasible? (NA for some epidemiological studies) Yes
Validity Questions
1. Was the research question clearly stated? Yes
  1.1. Was (were) the specific intervention(s) or procedure(s) [independent variable(s)] identified? Yes
  1.2. Was (were) the outcome(s) [dependent variable(s)] clearly indicated? Yes
  1.3. Were the target population and setting specified? Yes
2. Was the selection of study subjects/patients free from bias? Yes
  2.1. Were inclusion/exclusion criteria specified (e.g., risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study? Yes
  2.2. Were criteria applied equally to all study groups? Yes
  2.3. Were health, demographics, and other characteristics of subjects described? Yes
  2.4. Were the subjects/patients a representative sample of the relevant population? ???
3. Were study groups comparable? Yes
  3.1. Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT) No
  3.2. Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline? Yes
  3.3. Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.) Yes
  3.4. If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis? N/A
  3.5. If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.) N/A
  3.6. If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")? N/A
4. Was method of handling withdrawals described? Yes
  4.1. Were follow-up methods described and the same for all groups? Yes
  4.2. Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate) and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%.) Yes
  4.3. Were all enrolled subjects/patients (in the original sample) accounted for? Yes
  4.4. Were reasons for withdrawals similar across groups? N/A
  4.5. If diagnostic test, was decision to perform reference test not dependent on results of test under study? N/A
5. Was blinding used to prevent introduction of bias? Yes
  5.1. In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate? Yes
  5.2. Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.) Yes
  5.3. In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded? N/A
  5.4. In case control study, was case definition explicit and case ascertainment not influenced by exposure status? N/A
  5.5. In diagnostic study, were test results blinded to patient history and other test results? N/A
6. Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described? Yes
  6.1. In RCT or other intervention trial, were protocols described for all regimens studied? Yes
  6.2. In observational study, were interventions, study settings, and clinicians/provider described? N/A
  6.3. Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect? ???
  6.4. Was the amount of exposure and, if relevant, subject/patient compliance measured? Yes
  6.5. Were co-interventions (e.g., ancillary treatments, other therapies) described? N/A
  6.6. Were extra or unplanned treatments described? N/A
  6.7. Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups? Yes
  6.8. In diagnostic study, were details of test administration and replication sufficient? N/A
7. Were outcomes clearly defined and the measurements valid and reliable? Yes
  7.1. Were primary and secondary endpoints described and relevant to the question? Yes
  7.2. Were nutrition measures appropriate to question and outcomes of concern? Yes
  7.3. Was the period of follow-up long enough for important outcome(s) to occur? ???
  7.4. Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures? Yes
  7.5. Was the measurement of effect at an appropriate level of precision? Yes
  7.6. Were other factors accounted for (measured) that could affect outcomes? ???
  7.7. Were the measurements conducted consistently across groups? Yes
8. Was the statistical analysis appropriate for the study design and type of outcome indicators? ???
  8.1. Were statistical analyses adequately described and the results reported appropriately? No
  8.2. Were correct statistical tests used and assumptions of test not violated? ???
  8.3. Were statistics reported with levels of significance and/or confidence intervals? Yes
  8.4. Was "intent to treat" analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a dose-response analysis)? N/A
  8.5. Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)? ???
  8.6. Was clinical significance as well as statistical significance reported? Yes
  8.7. If negative findings, was a power calculation reported to address type 2 error? Yes
9. Are conclusions supported by results with biases and limitations taken into consideration? Yes
  9.1. Is there a discussion of findings? Yes
  9.2. Are biases and study limitations identified and discussed? Yes
10. Is bias due to study's funding or sponsorship unlikely? Yes
  10.1. Were sources of funding and investigators' affiliations described? Yes
  10.2. Was the study free from apparent conflict of interest? Yes